bluuebunny/crossref_metadata_2025 huggingface dataset:
Continuous reduction of heart rate by ivabradine cannot prevent deterioration of cardiac function in middle‐aged rats with a large myocardial infarction
Authors: McCooey Daniela , Bogatyryov Yevgen , Tomanek Robert J , Dedkov Eduard I
Published: June 2021

A large myocardial infarction (MI) initiates progressive structural remodeling that leads to a decline in left ventricular (LV) performance. Four weeks of heart rate reduction (HRR) in post‐MI rats by ivabradine (IVA), a selective inhibitor of the pacemaker If current, attenuated LV systolic dysfunction without modifying cardiac remodeling. However, continuation of this beneficial effect after an extended period of IVA treatment remains to be confirmed. Accordingly, we designed our study to determine whether prolonged HRR by IVA alters post‐MI LV remodeling and functional performance. A large MI (>; 50% of the LV free wall) was induced in 12‐month‐old male Sprague‐Dawley rats by ligation of the left coronary artery and the rats were then assigned in two experimental groups. In the first group, rats were treated with IVA i.p. via osmotic pumps in a dose of 10.5 mg/kg/d for 3 months (MI+IVA), while in the second group, rats received placebo (MI). Three months after MI, the infarct size and its composition, LV chamber dimensions and a degree of myocardial hypertrophy were comparable between MI and MI+IVA rats. At the same time, IVA‐treated rats showed a similar reduction in LV ejection fraction compared to untreated MI rats (0.32±0.02 vs. 0.30±0.02, respectively). Our data demonstrate that a prolonged period of HRR by ivabradine cannot prevent deterioration of LV systolic performance in middle‐aged rats after a large MI.

Grant Funding Source: Internal Award from the NYCOM Office of Research (E.I. Dedkov) and Research Grant from the IRI Servier (R.J. Tomanek)
Chick embryo particulate matrix involved in cardiac mesenchyme formation does not include BMP‐2
Authors: Abd‐Elhamid Tarek Hamdy , Conway Marianne L. , Sinning Allan R
Published: June 2021

Early heart development involves transformation of endothelial cells in the atrioventricular (AV) canal and outflow tract (OT) to mesenchymal cells, a process called endothelial mesenchymal transition (EMT). In these regions, EMT is initiated by factors localized in the particulate form of matrix secreted by the myocardium. This particulate matrix can be extracted using EDTA. Only the EDTA‐soluble extract and myocardial conditioned media (MyoCM) can replace the myocardium and initiate EMT. Since bone morphogenetic protein 2 (BMP‐2) has also been reported to play a role in EMT, we asked whether BMP‐2 is a part of the particulate matrix. To answer this question, we double immunostained stage 15–16 chick embryo sections with hLAMP‐1 and BMP‐2 antibodies. We found that BMP‐2 signals did not colocalize with hLAMP‐1 stained particles in the heart. In addition, we immunoprecipitated the particulate matrix using hLAMP‐1 antibody and examined the hLAMP‐1 bound fraction of EDTA extract and MyoCM for the presence of BMP‐2 using Western blot analysis. We found no association of BMP‐2 with the hLAMP‐1 bound fraction of the EDTA extract and MyoCM. Furthermore, we immunostained the cryopreserved EDTA extracted hearts with hLAMP‐1 and BMP‐2 antibodies. We observed that the vast majority of the hLAMP‐1 stained particles were extracted with EDTA, however, BMP‐2 signals were still present in these hearts. Our results indicate that BMP‐2 is not a component of the particulate matrix. These results contradict previous reports that BMP‐2 initiates the EMT process and question the importance of BMP‐2 in early stages of EMT.

Grant Funding Source: none
Ultrastructure and Immunohistochemical Analyses of a Regenerative Myocardium
Authors: Cox Serenity Dawn Stokes , Washburn Lyndsey , Pardhanani Pooja , Price Robert , Evans‐Anderson Heather
Published: June 2021

Ciona intestinalis is a well‐characterized invertebrate model system presenting many advantages in the study of cardiac biology. In contrast to most vertebrates, Ciona myocardium regenerates throughout the life cycle, making Ciona an interesting candidate for studying heart formation and repair mechanisms. In order to evaluate the cardiac regenerative response to injury, Ciona hearts were ligated and fixed for TEM or immunohistochemical (IHC) analysis following 24 or 48 hour recovery periods. TEM analysis reveals the rejection of degenerated cells and stark changes in mitochondrial organization following injury. A marked thickening of a currently unidentified matrix in the lumen is also evident. Key morphological features indicating myocardial regeneration include undifferentiated cell population in myocardium as well as cardiomyocyte differentiation and division. Currently, histological stains with H&E and pentachrome are being used to identify the extracellular matrix composition and to visualize changes from injury. In addition, IHC studies will determine apoptotic, proliferative, and myofibril changes incurred by injury. This study is the first to document an anatomical regenerative response to myocardial injury in Ciona. Understanding the process of myocardial regeneration in Ciona will help to elucidate the regulatory mechanisms of cardiac myocytes in higher order organisms.

Grant Funding Source: NIH AREA Evans‐Anderson
Intranasal Delivery of a Cystatin C‐peptide as Therapy for Alzheimer's Disease
Authors: Shaw Phyllis , Zhang Xu
Published: June 2021

Our long‐term goal is to develop therapies for Alzheimer's disease based on CysC‐AβBP, a peptide known to bind amyloid beta peptides and prevent their oligmerization in vitro. We hypothesize that this intervention should reduce amyloid beta diffusible ligands and plaque deposition, as well as improve memory in AD mice. Our results indicate that CysC‐AβBP binds specifically and preferentially to purified Aβ42 (a scrambled peptide does not bind Aβ42), and that in 2 AD mouse models, one with amyloid plaques and oligomers (oAβ) (TgCRND8) and one with oAβ only (APPE693Q), CysC‐AβBP co‐localizes with Aβ deposits in the neocortex and hippocampus when incubated with brain sections in vitro. In our in vivo studies, 2.7 and 4 month old CRND8 AD mouse littermates were treated intranasally with 20 μg of CysC‐AβBP or scrambled AβBP peptide, twice weekly for 3 weeks. The brain sections from untreated and treated AD were treated with amyloid beta specific antibody 6E10. The brains of AD mice with no treatment or scrambled peptide treatment had a large accumulation of amyloid beta aggregates and plaque in the neocortex and hippocampus. In contrast, we observed a profound reduction in β‐amyloid aggregates and plaques in the neocortex and hippocampus of CysC‐AβBP treated AD mice. Our results suggest that CysC‐AβBP reduces amyloid aggregates and plaque in animals developing AD and in animals with established AD.
Transforming growth factor betas (TGF‐βs) are novel extrinsic determinants in the induction and survival of mouse rostral hindbrain serotonergic neurons
Authors: Roussa Eleni , Gaedicke Simon , Krivokuca Dragan
Published: June 2021

Cell fate decisions along the dorsoventral and anterior‐posterior axis of the neural tube are dictated by diffusible signals from signaling and organizing centers. Development of rostral hindbrain 5‐HT neurons depends on Shh, FGF8, and FGF4. However, phenotype analyses from gene ablations studies have demonstrated that additional determinants should be involved in the specification of 5‐HT neurons. In our previous work we have shown that TGF‐βs are required for induction and survival of mesencephalic dopaminergic neurons. Here, we have investigated the impact of TGF‐βs in the differentiation and survival of mouse rostral hindbrain serotonergic neurons. Analysis of the Tgfβ2−/− mutant mice showed decreased number of 5‐HT neurons at E12.5 and E18.5. In vitro, treatment of dissociated neurospheres from rostral hindbrain with anti‐TGF‐β function blocking antibody also decreased the number of 5‐HT immunoreactive cells. Moreover, in ovo treatment of chick embryos between E6 to E10 significantly decreased the number of 5‐HT neurons, accompanied with an increase in the number of TUNEL positive cells. These data demonstrate that TGF‐βs are required for differentiation of hindbrain progenitors into serotonergic neurons and for survival of differentiated 5‐HT neurons and introduce TGF‐β2 as a novel extrinsic determinant in the molecular network underlying establishment of serotonergic cell fate.
Central Projections of the lagena in fixed chick embryos
Authors: Mahmoud Amany Refaat , Reed Caitlyn T. , Maklad Adel
Published: June 2021

Perception of linear acceleration is the function of the utricle and the saccule in mammals, whereas non mamalian vertebrates possess a third otolith endorgan, the lagena. Based on the findings of neuroanatomical tracing studies, the afferents of the lagena of the pigeon projected to the vestibular nuclei, and also to the cochlear nuclei, different functions were ascribed to the lagena of arboreal birds, as hearing and equilibrium. In the current study, we asked whether there is a difference in the central projections from the lagena in an arboreal from a terrestrial bird. To answer this question, the sensitive lipophilic tracer, Neuro veu red was selectively implanted in the sensory epithelium of the lagena of paraformaldehyde fixed chicken embryos. We found that all vestibular nuclei, the tangential nucleus, and the uvula of the cerebellum received lagenar projections. We also showed colossal fibers and tuft like terminals in the tangential nucleus. No lagenar terminals were found in the cochlear nuclei. In the cerebellum, the labeled terminals were found in the cerebellar nuclei, and the uvula. Double labeling was employed in the lagena, using Neuro veux red, and in the basilar papilla using Neuro veux maron. The Neuro veux red travelled to the vestibular nuclei, and the Neuro veux maron travelled to the cochlear nuclei.

Conclusion

our findings suggest that since the lagena in the chick projects to the vestibular nuclei and cerebellum and not to the cochlear nuclei, thus it is unlikely to have a role in hearing, as that suggested in the pigeon, rather, it may play an important role in equilibrium.

Grant Funding Source: none
Persistently increased hippocampal PKMζ expression correlates with spatial long term memory maintenance
Authors: Hartley Benjamin R , Tsokas Panayiotis , Hsieh Changchi , Fenton Andre A , Marquez Samuel , Sacktor Todd C
Published: June 2021

The constitutively active, atypical protein kinase C isoform PKMζ has been strongly implicated in the maintenance of long‐term memory (LTM) in various animal models (Sacktor TC, Mol Brain 18;5(1): 31 [2012]). PKMζ is also involved in spatial LTM, and its necessity for maintaining memory has been demonstrated days and even weeks after memory formation (Pastalkova E, et al., Science 313(5790): 1141–1144 [2006]). Here, we show that PKMζ expression is significantly increased in the hippocampus after memory formation, specifically in the stratum radiatum, stratum lacunosum‐moleculare, and CA1 pyramidal cell layers. Adult male wild‐type mice received 4 active place avoidance training trials, at 1 hour intervals, for 2 consecutive days. Their retention was tested 1 month later. Their brains were harvested, fixed in 4% paraformaldehyde, and sectioned into 40 μM slices. PKMζ localization was analyzed using immunohistochemistry (IHC) and confocal microscopy. Western blots of hippocampal homogenates were also performed. We show that PKMζ levels increased in the aforementioned areas, compared to unconditioned animals. Additionally, the level of PKMζ expression by Western blot and IHC correlates with LTM performance. Thus, we argue that PKMζ protein is a physical substrate of the engram and is responsible for the maintenance of LTM.

Grant Funding Source: NIH Grant R01DA034970
The involvement of Programmed Cell Death (PDCD5) in the regulation of apoptosis in cerebral ischemia/reperfusion injury
Authors: Zhou Changman , Chen Chunhua , Jiang Zhao , Yan Junhao , Chen Yingyu
Published: June 2021

Aim

We found for the first time that Programmed Cell Death 5 (PDCD5) plays an essential role in brain ischemic injury in a rat focal cerebral ischemia model. We hypothesized that down‐regulating PDCD5 can protect the brain from ischemic damage by inhibiting PDCD5 induced apoptotic pathway.

Methods

105 Sprague‐Dawley male rats weighing 280 to 300 g were randomly assigned to the following five groups: Sham surgery (n=20), MCAO (n=24), MCAO treated with recombinant human PDCD5 (rh PDCD5) (n=20), MCAO treated with control siRNA (n=20), MCAO treated with PDCD5 siRNA (n=20).

Results

PDCD5 siRNA reduced the infarct volume, improved neurological deficits and reduced Evans blue extravasation; over‐expression of PDCD5 protein using recombinant human PDCD5 (rhPDCD5) had the opposite effect. But unfortunately, changed PDCD5 expression had no significant effect on mortality. Immunohistochemistry and western blot demonstrated PDCD5 siRNA decreased expressions of key pro‐apoptotic proteins such as p53, Bax/Bcl‐2 and cleaved casepase‐3 in the infarcted areas, whereas PDCD5 overexpression attenuates cell apoptosis. Double fluorescence labeling showed PDCD5 positive immunoreactive materials were partly colocalized with MAP2, GFAP, p53 and caspase‐3 in the injured cerebral cortex.

Conclusion

PDCD5 induced apoptosis and over‐expression of PDCD5 is harmful to the ischemic neurons in vivo. Inhibition of PDCD5 may be protective via reducing the apoptotic‐related protein such as p53, Bax and caspase‐3. This observation may have potential for the treatment of ischemic cerebral stroke.
Expression of Mineralocorticoid and Glucocorticoid receptors in Pre‐autonomic Neurons of the Rat Paraventricular Nucleus
Authors: Chen Jian , Gomez‐Sanchez Celso E. , May Paul J. , Gomez‐Sanchez Elise P.
Published: June 2021

Plasma aldosterone levels that are inappropriately high for the sodium intake increases sympathetic nervous system (SNS) activity and the hypertension and increased SNS activity in experimental mineralocorticoid‐salt excess is abrogated by lesions of the paraventricular nucleus (PVN) or antagonists of the mineralocorticoid receptors (MR). It is uncertain whether physiological concentrations of aldosterone bind MR in the PVN; corticosterone and cortisol bind both MR and glucocorticoid (GR) receptors at physiological concentrations and MR and GR influence each other at transcriptional and functional levels. The retrograde tracer Fast Blue was injected into the IMLCC at the level of T4 and T10 of spinal cord after laminectomy to locate preautonomic neurons in the PVN. Immunofluorescent histochemistry was done in frozen modified STF‐fixed brains using specific anti‐bodies against the MR and GR to determine colocalization of these two receptors within pre‐autonomic neurons. Three different pseudo‐colors were assigned by Image‐Pro for better visualization. Expression of GR and MR varied in different parts of the PVN. Most colocalization was in mid‐PVN parvocellular neurons. Very few magnocellular pre‐autonomic neurons were found to express GR and/or MR. Adrenocortical steroids could have direct effects on sympathetic activity by binding MR and/or GR in pre‐autonomic neurons of the mid‐PVN. This limited area of expression may explain the inconsistent results of studies on the effect of PVN microinjections of MR and GR agonists and antagonists on sympathetic drive.

Grant Funding Source: VA BLRDB‐ORD 1018X007080, NIH/HLBI RO1HL0272, and NIH/NCRR COBRE (Center for Psychiatric Neuroscience)
Conformational analysis of processivity clamps demonstrates that tertiary structure does not correlate with structural dynamics
Authors: Nevin Philip , Fang Jing , Engen John R , Beuning Penny J
Published: June 2021

Chromosomal replicases faithfully catalyze the polymerization of thousands of DNA bases each second without dissociating from DNA. Their remarkable processivity is attributed to sliding clamps, which are structurally conserved proteins that encircle DNA and tether the DNA polymerase to the DNA template. Here we report a comprehensive comparative study of the conformational dynamics of sliding clamps from a variety of sources ranging from phage to humans. We have probed the structural dynamics of these proteins in solution using hydrogen exchange mass spectrometry and show that sliding clamps from different species show a wide range of dynamic behavior despite highly similar three‐dimensional structures. In addition to large variations in the degree of structural fluctuations, several of the clamps undergo local unfolding events with half‐lives ranging from five minutes to several hours. Furthermore, the domains within each clamp exhibit different dynamics despite their obvious structural symmetry. Our observations reveal that the human clamp PCNA is more dynamic than other eukaryotic clamps, consistent with thermal denaturation experiments.

We acknowledge generous financial support from a New Faculty Award from the Camille & Henry Dreyfus Foundation, the American Cancer Society, the NSF, the NIH, and a research collaboration with Waters Corporation.
Investigating the properties of a repair replication‐fork in the budding yeast Saccharomyces cerevisiae
Authors: Anand Ranjith Prasad , Haber James E
Published: June 2021

Genome instability is the hallmark of several human diseases including cancer. The FoSTeS (Fork Stalling and Template Switching) model – also termed Microhomology‐Mediated Break‐induced Replication (MM‐BIR) – posits that the fork originating from a stalled or collapsed fork can trigger genome instability by means of template switching of the repair‐fork. In order to examine MM‐BIR in detail, we created a system in S. cerevisiae in which a single double‐strand break induced by HO endonuclease can be repaired by a two step process that involve 1) strand invasion with a donor sequence followed by 2) template switch located at about 50 kb away generating a functional URA3 gene as part of a complex nonreciprocal translocation. Our results show that when the sequences are 100 % homologous, these events occur at a frequency of about 2 × 10−3. The presence of sequence heterology had distinct affect on the two steps; heterology severely restricted strand invasion by about four orders of magnitude, while template switch was less affected. For the first time, our results reveal two important features distinguishing canonical strand invasions from template jumps: 1) template jumps were less restrained by heterologies than strand invasions providing evidence for their distinct nature and 2) invasions preferred the use of the very 3′ end whereas template jumps showed no preferred sequences, suggesting repeated strand dissociation and reformation as a hallmark of repair forks. Sequencing of junctions of recombined sequences that shared no significant homologies showed that such events utilized sequences ranging from 3 bp micro‐homologies to no homologies.
Identification and characterization of novel small molecule inhibitors of the human Y‐family DNA polymerases
Authors: Ketkar Amit , Zafar Maroof K , Maddukuri Leena , Penthala Narsimha R , Crooks Peter , Coggins Grace E , Lloyd R. Stephen , Eoff Robert L
Published: June 2021

Specialized Y‐family DNA polymerases are responsible for bypass of replication blocks, such as DNA adducts or G‐quadruplexes. Misregulation of Y‐family members is associated with many tumor types. They are highly successful in performing their role due to certain unique structural and biochemical properties. These same properties, however also make these enzymes more error‐prone and capable of producing mutations. This phenomenon can be coopted by tumor cells as a mechanism to survive chemotherapeutic and ionizing radiation treatments. Thus, these enzymes represent potential targets for adjuvant cancer therapies. Recently, the inhibitor of 5‐lipoxygenase activating protein, MK886, was discovered as one of the more potent inhibitors of a specialized DNA polymerase hpol κ. In our study, we investigated the specificity and mechanism of inhibition of MK886 against other human Y‐family DNA polymerases, as well as a model B‐family DNA polymerase. We have expanded our studies to include inhibitors that were identified by screening a targeted library of compounds synthesized by the laboratory of Prof. Peter Crooks (UAMS). Kinetic analyses and in silico docking studies were used to determine the mechanism of polymerase inhibition by these novel compounds. Our results provide a framework for developing potent inhibitors of translesion DNA polymerases.

Supported in part by USPHS R00 GM084460 (R.L.E.)
A role of heterogeneous nuclear ribonucleoprotein A/Bs in telomere maintenance
Authors: Wang Tzu‐Chien V , Huang Pei‐Rong
Published: June 2021

Several members of the heterogeneous nuclear ribonucleoproteins (hnRNPs) family are thought to play roles in telomere maintenance because they are known to bind to telomeric DNA and to TERRA RNA, which is now identified as an integral component of telomeric heterochromatin. We have previously shown that the members of hnRNP A/B family are the most abundant proteins in the nuclear extracts that bound specifically to G‐strand telomeric DNA. To address if members of hnRNP A/B family are essential components of a functional telomere (i.e., does hnRNP A/Bs play a structural role) or are involved in telomere length regulation, we investigated the biological effects of knocking‐down hnRNP A1, A2/B1 or A3 in OEC‐M1 cells. Transient depletion of single hnRNP A1, A2/B1 or A3 did not affect cell growth. Stable clones that were singly knock‐down for hnRNP A1, A2 or A3 displayed normal cell proliferation and their telomere lengths were stable over 100 population doublings. Interestingly, while double‐knockdown of hnRNP A1&A3 produced a moderate inhibition of cell growth, double‐knockdown of hnRNP A1&A2 completely inhibited cell growth and was accompanied with the induction of apoptosis. These results suggest that there is a functional overlap among hnRNP A1, A2, and A3. Works are in progress to determine whether the anti‐proliferation effects of double‐depleting hnRNP A/Bs may result from telomere dysfunction.
Purification and characterization of a metal‐dependent regulator protein from Thermobifida fusca
Authors: Granger Joseph Benjamin , Nguyen Tuan , Lu Zeyu , Ferguson Jordan B. , Maria Peter Santa , Novak Walter R.P.
Published: June 2021

Iron dependent regulators are proteins found in a variety of pathogenic bacteria that cause diseases such as tuberculosis and diphtheria. We recently characterized a crudely purified metal‐dependent regulator from Thermobifida fusca, and demonstrated binding to the DtxR tox operator consensus sequence. The regulator from T. fusca has a longer N‐terminal helix than homologs such as DtxR. Conformational changes have been shown to occur upon metal binding in homologous proteins, and it has been suggested that this helix is important in metal and DNA binding. We have now purified the protein to greater than 90% purity as judged by SDS‐PAGE, and also report the Tf‐IdeR protein binds the EntS operator sequence from its own genome. Additionally, computational analysis of the N‐terminus indicates Ile7 may be important for metal‐dependent DNA binding. This research is supported by the Wabash College Haines Biochemistry Fund.
The potential role of Simian Virus 40 polyA sequence (SV40 polyA) to mediate stress‐inducible mRNA translation
Authors: Wong Naisum , Lui Thomas Wan
Published: June 2021

Viral infection of a cell is often associated with the development of cellular stress. Although the inhibition of global mRNA translation is a common cellular response to stress, many viruses are still able to exploit the protein synthesis machinery of the host cells for production of viral proteins. In this study, we examined if certain sequence element in the SV40 viral genome may enable synthesis of viral proteins to take place despite the presence of cellular stress. The pEGFP‐c1 expression plasmid that incorporates the SV40 polyA sequence in its transcript was used as a model system for this study. We found that the expression of pEGFP‐c1 in several mammalian cell lines could be markedly enhanced by the presence of stress inducers such as arsenite, cadmium, and thapsigargin, all of which were shown to result in eIF2‐alpha phosphorylation and inhibition of global protein synthesis. The co‐treatment of cells with cycloheximide abolished the stress‐induced increase in GFP expression. The deletion of the 5′ 1–120 nucleotides of SV40 polyA markedly diminished stress‐induced increase of GFP expression. However, the expression of GFP using the pcDNA3 plasmid that does not have the SV40 polyA in its transcript was not altered in the presence of stress inducers. These results suggest the interesting idea that SV40 polyA play a hitherto unrecognized role in viral gene expression.
The effect of N‐butyl‐deoxynojirimycin on the structure, function and trafficking of intestinal glycoproteins
Authors: Amiri Mahdi , Naim Hassan Y.
Published: June 2021

N‐butyl‐deoxynojirimycin (NB‐DNJ) is a synthetic analogue of D‐glucose which is used for treatment of the Niemann‐Pick Type C and Gaucher diseases. This drug is consumed orally and in most of the cases gives rise to gastrointestinal intolerances similar to congenital carbohydrate intolerance disorders, strongly suggesting that the activities of the disaccharidases in the intestine are affected. We have investigated the role of NB‐DNJ on the major disaccharidases of the intestinal epithelium. Our findings show that an immediate effect of NB‐DNJ is its interference with carbohydrate digestion in the intestinal lumen via reversible inhibition of disaccharidases that cleave α‐glycosidically linked carbohydrates. NB‐DNJ elicits also long term effects on the biosynthesis and processing of intestinal glycoproteins. Thus it reduces the trafficking rates of these proteins, since it interferes with their N‐glycosylation in the ER. These altered glycoforms lose partially their enzymatic function, yet their folding pattern does not grossly change as assessed by the similar proteolytic pattern of the various glycoforms. Therefore a reduction in the activities of brush border enzymes in the apical membrane of intestinal cells occurs due to the combinatorial effects of NB‐DNJ on the trafficking and N‐glycan pattern as well as the immediate inhibition of the enzymes of the brush border membrane.
Unravelling the specificity in the ubiquitin system
Authors: Komander David
Published: June 2021

Eight different types of homotypic ubiquitin chains exist, and different ubiquitin linkages have been associated with distinct cellular processes. Enzymatic assembly systems to generate ubiquitin chains are available for Lys11‐, Lys48‐, Lys63‐ and Met1‐linked chains, and we have recently established enzymatic assembly systems for Lys6‐linkages. Chemical biology methods have made the remaining chain types linked via Lys27, Lys29 and Lys33 available.

Access to all ubiquitin linkages allows a comprehensive analysis of linkage‐specificity in deubiquitinases. We have recently analysed the entire human family of OTU deubiquitinases, to show that each member comprises distinct linkage preference. Structural and biochemistry studies reveal four distinct mechanisms of linkage specificity, allowing the design of enzymes with novel specificity by mutagenesis of residues near the active site. Linkage‐specific OTU deubiquitinases can be used as ‘restriction enzymes’ in ubiquitin chain sequencing applications, to allow quick assessment of linkage type and topology on a protein.
Inhibition of Endosome Tubulation with Phospholipase A2 Antagonists Prevents Transport of HIV‐1 across Mucosal Vaginal Epithelial Cells
Authors: Kinlock Ballington , Wang Yudi , Liu Bindong
Published: June 2021

The mechanism by which HIV‐1 navigates the vaginal milieu en route to cause systemic infection is not fully understood. Vaginal epithelial cells are capable of taking up HIV‐1 and passing it along to HIV‐1 susceptible cells through trans‐infection. Trans‐infection is believed to occur in part through transcytosis which may utilize several different trafficking pathways. Data depicting the exact cellular pathway that the virus exploits to traverse vaginal epithelial cells remains incomplete. Our data shows that HIV‐1 enters vaginal epithelial cells through endocytosis which is interrupted by endocytic inhibitors. In addition, we demonstrate the intracellular trafficking pathway that HIV‐1 exploits to get across vaginal epithelial cells. After allowing equal amounts of HIV‐1 to enter vaginal cells; our data shows that reduction of endosome tubulation inhibited the release of HIV‐1. Inhibition of this pathway caused an accumulation of HIV‐1 within the cells. We also demonstrate that expression of the dominant negative Rab‐11 protein had a similar effect where HIV‐1 release from these cells was inhibited compared to our transfected control. We conclude that passage of HIV‐1 through vaginal epithelial cells is dependent on endosomal tubule formation and exploits the Rab‐11 dependent recycling pathway.

Support provided by NIH/NHLBI 5T32HL007737–19 and NIH/NIGMS 1SC1GM089269
Combinatorial post‐translational modifications of RNA polymerase II
Authors: Lothrop Adam , Kulla Daniel , Lin Yu‐Shan , Fuchs Stephen
Published: June 2021

RNA polymerase II (RNAP II) is responsible for transcription of the majority of mRNA in eukaryotes. The C‐terminal domain (CTD) of RNAP II contains multiple types of post‐translational modifications (PTMs), which facilitates the interactions between a large number of transcriptionally‐associated factors and the CTD itself. Temporal regulation of these binding events is orchestrated by a dynamic arrangement of PTMs. The prolyl isomerase Pin1 impacts the CTD phosphorylation state, demonstrating cross‐talk between these two types of PTMs. To investigate the relationship between these modifications, we have constructed a library of CTD peptides containing varying combinations of phosphoserine and a proline analog that is biased towards the cis conformation. With the noted importance of CTD phosphorylation, we used these peptides in array based assays to determine the effects of proline isomerization on CTD kinase activity. Assay data is combined with a computational approach, utilizing molecular dynamics simulations to investigate PTM effects on CTD conformation. The data from this study provides a detailed portrait of the complex interplay of PTMs in regulating important interactions of the CTD with other protein factors during transcription. Supported by Tufts University startup funding to S.M.F.
Yeast Rab5 signaling in endolysosomal trafficking and MVB biogenesis
Authors: Merz Alexey J. , Paulsel Andrew L. , Russell Matthew R.G. , Nickerson Daniel P.
Published: June 2021

Small G proteins of the Rab5 family control traffic through early compartments of the endolysosomal system. Saccharomyces cerevisiae, like mammals, contains three Rab5 paralogs: Vps21, Ypt52, and Ypt53. We present experimental analyses of the roles of the overlapping and distinct functions of these Rabs, and we analyze the proteins that activate and terminate their signaling activities. We demonstrate that Rab5 signaling is essential for the biogenesis of morphologically normal endosomal multivesicular bodies (MVBs) and for the correct targeting of MVB cargos en route from the Golgi or plasma membrane. In addition to the known nucleotide exchange factor Vps9, we identify Muk1 as a second partially redundant activator of Rab5 family members, and we present biochemical and functional studies of the relative contributions of Vps9 and Muk1. As with the Rab5 paralogs themselves, Vps9 and Muk1 are essential for correct sorting of cargo into MVB intraluminal vesicles. Finally, we demonstrate that signaling by Vps21, the most biologically important of the three yeast Rab5 family members, is selectively terminated by the Gyp3 Rab GAP.
Substrate promiscuity of β‐Ketoacyl ACP Synthase III (KASIII): Understanding the structural basis for functional diversity of KASIII enzymes
Authors: Garg Shivani , Yandeau‐Nelson Marna D. , Nikolau Basil J.
Published: June 2021

β‐Ketoacyl ACP Synthase III (KASIII) catalyzes the Claisen condensation reaction that initiates fatty acid synthesis in bacteria and plants. KASIII from diverse sources can exhibit a broad range of substrate specificities. For example, E. coli KASIII uses straight chain acyl‐CoA substrates, whereas the B. subtilis homologs, KASIIIa and KASIIIb, use both straight and branched chain acyl‐CoA substrates. The structural basis for KASIII functional diversity remains obscure. Based on the hypothesis that the KASIII substrate specificity is determined by the rotamer conformation of a conserved Phe residue, we mutated several residues which are located close to this residue and can affect its rotamer conformation. The interactions of the mutated enzymes with their acyl‐CoA substrates (either straight or branched chain) were studied using Saturation Transfer Difference NMR. STD NMR data indicate that mutations of two residues in E. coli KASIII to the corresponding residues in B. subtilis KASIIIa increase straight and branched substrate binding by ~80% and ~66%, respectively. The corollary mutations in B. subtilis KASIIIa reduced the binding affinity for these substrates. Hence, this study has identified two key residues that govern the KASIII substrate specificity. This knowledge will enable engineering of KASIII biocatalysts with novel substrate specificities, which may have numerous downstream applications.
Effect Of 21‐Benzylidene Digoxin On Cancer Cells: A Cardenolide With No Inhibitory Effect On Na,K‐ATPase Activity
Authors: Pessoa Marco Túlio Correa , Neves Luiza D.R. , Rocha Sayonarah C. , Alves Silmara L.G. , Villar José Augusto F.P. , Silva Luciana M , Oliveira Soraya M.F. , Taranto Alex G. , Gomes Isabella V. , Santos Fábio V. , Quintas Luis Eduardo M. , Noel François G. , Santos Hérica L. , Barbosa Leandro A
Published: June 2021

Recently, new data began to consider cardiac glycoside as a potential antitumor drug. We synthesized 21‐benzylidene digoxin (21‐BD) through an aldol condensation of digoxin lactone ring. Molecular docking was performed to assess the main interactions between cardiac glycosides and Na,K‐ATPase, demonstrating a more stable binding energy for 21‐BD, compared with digoxin. Therefore, we used HeLa and RKO cancer cell lines after their exposure to cardiac glycosides to asses the cell viability as to cytotoxicity and proliferative ability. On HeLa cells, digoxin presented an IC50 = 1,47 μM, whereas 21‐BD presented an IC50 = 56,16 μM. Nevertheless, on RKO cells, digoxin presented an IC50 = 0,46 μM and 21‐BD presented an IC50 = 54,36 μM. Cometa assay showed DNA damage upon 20 μM of both cardiac glycosides. The treatment of cells with 21‐BD for 48h caused an increase of Na,K‐ATPase activity, whereas digoxin caused a decrease on enzyme activity. However, 21‐BD did not cause inhibition of Na,K‐ATPase of a membrane preparation from rat cerebral hemisphere. In attempt to avoid the 21‐BD effect though estrogen receptor, the pre‐treatment with tamoxifen did not interrupted the DGB1 effect. Thus, these data demonstrate that this new glycoside has a different mechanism of action then digoxin, and it can be an important tool to study the enzyme.

Supported by: FAPEMIG, CNPq and CAPES
Kinetic studies of synthetic argininal thiosemicarbazone containing inhibitors of cathepsin B
Authors: Wen Lisa , Yelma Niranjan Reddy , Lucas Joseph , Huang Jenq‐Kuen , McConnell Rose
Published: June 2021

Cathepsin B, a lysosomal cysteine protease, plays roles in diverse cellular processes including protein degradation, antigen processing, and apoptosis. It has been implicated in many diseases such as cancer invasion and metastasis, inflammation, and Alzheimer's Disease. Thus, cathepsin B is a promising target for the development of therapeutic agents for treatment of many types of cancer and Alzheimer's Disease. Previously prepared synthetic argininal thiosemicarbazone containing inhibitors were evaluated for their ability to inhibit recombinant human cathepsin B by determining their IC50 values. The studies were performed by colorimetric enzyme inhibition assay using N‐Carbobenzoxy‐L‐Phenylalanyl‐Arginine‐4‐nitroanilide hydrochloride as the chromogenic substrate. Kinetic properties of four potential inhibitors were characterized. Funding provided by National Cancer Institute at NIH (Grant No. 3R15CA086933–04 and 3R15CA086933–04A2S1) and Western Illinois University.
Role of Primary Structure Variation in Structure and Function of Calcineurin
Authors: Cook Erik , Dunlap Victoria , Creamer Trevor
Published: June 2021

A. fumigatus is one of the leading causes of invasive fungal infections in solid organ transplant and adult stem cell transplant recipients. It is estimated that over 20% of Americans are infected with T. gondii. T. gondii is life threatening in immunocompromised patients. Here, we study the enzyme calcineurin that is critical for the virulence and pathogenicity of these organisms. CaN is activated when calcium‐loaded calmodulin (CaM) binds the regulatory domain (RD) of the A subunit. Upon binding to CaM, the RD of human calcineurin transitions from a disordered state to an ordered state, gaining alpha helical structure. Research done in our lab has determined that the C‐terminus of the RD forms a helix (distal helix) that is necessary for the normal function of human CaN. In A. fumigatus and T. gondii, the sequence for this helix is deleted. A. fumigatus and T. gondii CaN contain modular insertions and deletions in the RD. A Ser/Pro rich region (SPRR) found in the RD that is unique to A. fumigatus is critical for the function of CaN and survival. The phosphorylation of this SPRR has also been shown to be critical in the pathogenicity of the organism. Investigations into the protein sequence of CaN of T. gondii show a unique insertion in the same area as the SPRR in A. fumigatus, but the function of this insertion is unknown. Thus, we are probing into how structure affects function through evolutionary variations.
The role of protein glycosylation in laccases from Lentinus sp.
Authors: LIU WEI‐CHUN , Hsu Chih‐An , Wang Nai‐Chen , Jeng Wen‐Yih , Wang Andrew H.‐J. , Shyur Lie‐Fen
Published: June 2021

Laccases are blue multicopper oxidases that can oxidize various organic and inorganic compounds by reducing O2 to water. Despite the decades’ research, the roles of protein glycosylation in laccases are still unclear. Here we report the crystal structure at 1.8 Å resolution of a native laccase (designated lcc4) from a white‐rot fungus Lentinus sp. in which two N‐glycosylated sites at Asn75 and Asn458 were identified. We observed the activity of native laccase was decreased significantly after α‐mannosidase digestion, suggesting the importance of glycosylation in laccase activity. To address the structural and functional role of glycosylation, we cloned laccase genes and created a number of mutant enzymes with the Asn→Asp mutation on predicted glycosylation residues, Asn75, Asn236, Asn399, and Asn458. The recombinant mutant enzymes expressed in Pichia pastoris all showed much lower activities compared to wild‐type counterpart. On the other hand, hyperglycosylation observed in the yeast expressed recombinant laccases may interfere the protein folding giving a decrease in the enzyme catalytic activity. This study provides some possible structural and functional roles of glycosylation in laccase enzymes. NSC‐97–3114‐P‐001–001, Taiwan
Profile of the liver sinusoidal endothelial cell in non‐alcohol fatty liver
Authors: Harris Edward N. , Gopalakrishnan Sandhya
Published: June 2021

Non‐alcoholic fatty liver disease (NAFLD) is an asymptomatic increasingly common disorder that affects liver metabolism. Liver sinusoidal endothelial cells (LSECs) regulate serum derived macromolecular exposure to hepatocytes and we asked what role LSEC could play in the pathogenesis of NAFLD. To test this, we used an animal model, Sprague‐Dawley rats, which were maintained on control and high fat (HF) diets for 8–9 weeks. The lipid burden in intact livers tissues and purified hepatocytes and LSECs were visualized by several methods specific for lipid staining. In the HF‐fed rats, there was an accumulation of lipid droplets in hepatocytes in contrast to the LSECs which did not show any lipid accumulation. LSECs isolated from control and fatty livers were plated on plastic plates and allowed to internalize 125I‐HA (radiolabeled hyaluronic acid) within the same day. LSECs from HF‐fed rats had reduced HA internalization levels compared to the control. We also observed that serum HA levels, free fatty acid, and alkaline phosphatase levels were increased in high fat diet fed rats, in contrast to alanine transaminase, triglycerides and cholesterol levels which remained the same as the controls. RNA from purified LSEC preparations was subjected to microarray analysis and revealed very different gene expression profiles between rats on control and HF diets.
Redox state as a master regulator of liver function
Authors: Nocito Laura , Yoo Elsia J , Corkey Barbara E
Published: June 2021

Objective

To determine if redox, as a regulator of metabolic and mitochondrial responses, controls the fate of fuels and liver function by generating reactive oxygen species (ROS).

Methods

Primary hepatocytes were treated with extracellular redox couples: cysteine/cystine (Cys/CySS), reduced/oxidized glutathione (GSH/GSSG), βhydroxybutyrate/acetoacetate (βOHB/Acoc) and lactate/pyruvate (L/P). ROS generation was measured using H2DCFDA. Endogenous glucose production and circulating redox metabolites were measured from cell incubation media using fluorescent methods. Oxygen consumption was measured using the XF24 extracellular analyzer.

Results

Redox couple induced oxidized conditions increased intracellular ROS generation. Endogenous glucose production, mitochondrial respiration, and ketone bodies and L/P secretions were also analyzed. Both endogenous glucose production and mitochondrial respiration decreased under oxidized conditions, whereas the ratios of ketone bodies (βOHB/Acoc) and L/P secretions increased.

Conclusions

We show that oxidized extracellular conditions induce ROS in primary hepatocytes. The extracellular oxidized milieu determined by βOHB/Acoc causes a decrease in the maximal respiratory capacity induced by the uncoupler FCCP. These effects are associated with a decrease in hepatocyte gluconeogenic capacity.
Mechanisms of acute lung injury: Programmed Cell Death Receptor (PD)‐1 ligation as a regulator of Angiopoietin‐2 expression
Authors: Lomas‐Neira Joanne , Monaghan Sean , Huang Xin , Chen Yaping , Ayala Alfred
Published: June 2021

PD‐1 is a co‐inhibitory protein on leukocytes and has been shown to negatively regulate T‐cell responses. PD‐1 ligand, PD‐L1, is expressed on immune and non‐immune cells including lung vascular endothelial cells (ECs). The basis for PD‐1's broad influence on immune response is unclear. Our laboratory has shown expression of PD‐1 contributes to mortality in our model of hemorrhage (Hem) priming followed by sepsis in the development of indirect acute lung injury (iALI). Initial data shows significant increase in PD‐L1 expression on ECs in the lungs and PD‐1 on Gr1+ cells in circulation following Hem. To the extent that this expression of PD‐1/PDL‐1 impacts lung EC dysfunction encountered during iALI, we assessed changes in EC growth factor Angiopoietin (Ang)‐2 in PD‐1−/− or PD‐L1−/−mice in our Hem/sepsis model for iALI. Ang‐2, released by activated ECs, is associated with vascular destabilization, inflammation and vascular permeability, and is significantly elevated in both patients and mouse models of iALI. Our data found that Ang‐2 was significantly reduced in both PD‐1−/− and PD‐L1−/− mice with iALI. However, MPO activity in lung tissue was significantly suppressed in PD‐1−/− mice, but not in PD‐L1 −/−. These data suggest PD‐1:PD‐L1 also play role(s) beyond the classic regulation of the leukocyte immune response; not only T‐cell mediation, but also in the innate response to inflammation as modeled in our iALI mice.
Endothelial cell IQGAP1 is an important regulator of the transendothelial migration of leukocytes
Authors: Sullivan David P , Muller William A
Published: June 2021

One of the key processes in the inflammatory response is the movement of leukocytes across the endothelium (transendothelial migration, TEM). We have shown that TEM requires a novel endothelial cell compartment, the Lateral Border Recycling Compartment (LBRC), which delivers requisite membrane proteins to surround the migrating leukocyte. In a recent proteomic screen to identify the components of the LBRC we discovered that IQGAP1 is specifically associated with it. Moreover, IQGAP1 is a critical factor in TEM. shRNA mediated knockdown of IQGAP1 expression in endothelial cells significantly impairs their ability to support TEM. In IQGAP1 knockdown cells, the targeted movement of the LBRC to the leukocyte is severely disrupted. This defect is rescued by re‐expression of full length IQGAP1. Using fluorescently tagged IQGAP1 constructs, we can follow IQGAP1 during TEM and have found that it moves with the LBRC to surround the migrating leukocyte. Furthermore, we are performing domain truncations of IQGAP1 to determine which of its many functional interactions are required for TEM. These data suggest that IQGAP1 is an important factor in the regulation of LBRC trafficking during TEM. This research was supported by F32 AI084454 to DPS and R21 HL102519, R01 HL046849, and R37 HL064774 to WAM.
Determining the presence of arsenic in chicken products using atomic absorption spectroscopy
Authors: Mercer Kevin J , Bradley Kathryn R , Burns Wendy L
Published: June 2021

Arsenic may enter the food supply because of its presence in contaminated soil and groundwater or from antibiotic use in livestock. One common antibiotic used for chicken is Roxarsone, an organoarsenical which has been used regularly in the past. While the Food & Drug Administration (FDA) has set standards for permissible levels of arsenic in meat products, the research exploring the concentrations of arsenic in chicken meat and eggs is limited. The purpose of this research was to test a method for the determination of trace amounts of arsenic in chicken products commonly sold at local supermarkets. Chicken breast, livers, and eggs were digested with concentrated acids and peroxides to release any potentially present arsenic from the tissues. Arsenic was then detected via atomic absorption spectroscopy and concentrations were determined from a standard curve. Preliminary results show detectable arsenic levels in some chicken breast samples. In vitro studies have demonstrated the inhibition of protein folding pathways from arsenic trioxides which also have implications in heart disease and various cancers. Recent legislature has helped to restrict Roxarsone use, but does not completely eliminate the presence of arsenic in chicken tissue.