bluuebunny/crossref_metadata_2025_split huggingface dataset:
Inhibition of Notch signalling induces extensive intussusceptive neo‐angiogenesis by recruitment of mononuclear cells
Authors: Djonov Valentin G. 
Published: June 2021

Objectiveto evaluate the effects of Notch inhibition on the vasculature using chick area vasculosa and MxCre Notch1(lox/lox) mice and to test to which extent this effect is mediated by ephrinB2/EphB4.Methods and ResultsInhibition of Notch signaling by Y secretase 1 induced significantly intussusceptive angiogenesis (IA) forming large immature capillary plexus. The latter it is characterized by 40% increased vascular density, pericyte detachment, increased transpermeability and recruitment of mononuclear cells to the sites of new formed transluminal tissue pillars (sign of IA). The process was dramatically enhanced when Notch inhibition was combined with the injection of chicken or mouse bone marrow‐derived mononuclear cells. Along with the morphological alterations of IA, we observed down‐regulation of ephrinB2 mRNA levels. Inhibition of ephrinB2 and EphB4 signaling induces pericytes detachment and significantly upregulation of VEGFR1 but without angiogenic response and recruitment of mononuclear cells. Remarkable, the chemotactic factors SDF‐1 and CXCR4 were up‐regulated only due to the Notch inhibition.ConclusionsOur results indicated that Notch inhibition disturbs vessel stability, induces extravasation of mononuclear cells of bone marrow origin by up‐regulation SDF‐1/CXCR4 chemotactic factors. Recruited cells contributed to formation of transluminal tissue pillars i.e. intussusceptive neo‐angiogenesis, triggering in this way the augmentation of the capillary plexus but without the accompanying vascular maturation and remodeling. .
Chronic ivabradine‐induced heart rate reduction does not modify collagen accumulation in myocardial infarction scar of middle‐aged rats
Authors: Bogatyryov Yevgen , McCooey Daniela , Tomanek Robert J , Dedkov Eduard I 
Published: June 2021

Heart rate reduction (HRR) by ivabradine (IVA), a selective inhibitor of the pacemaker If current, for 4 weeks after myocardial infarction (MI) was associated with the reduced collagen accumulation in surviving left ventricular (LV) myocardium. Although this effect is considered to benefit LV performance, the decrease in fibrillar collagen (FC) can diminish a tensile strength of the scar. Therefore, we designed our study to determine whether chronic HRR with IVA affects a structural composition of the MI scar. 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). After three months, the fractional volume of FC was determined in the myocardium of LV free wall and the scar. The scar size and thickness were comparable between MI and MI+IVA rats (63.1±2.7% vs. 59.6±1.8% and 0.93±0.09mm vs. 0.90±0.05mm, respectively). Although, there was a significant reduction of FC in LV free wall of IVA‐treated rats (5.1±0.5% vs. 7.8±0.8%, p≤0.01) the content of FC was comparable in scars of MI and MI+IVA rats (71.8±3.9% vs. 71.7±6.9%, respectively). Our data show that chronic IVA‐induced HRR does not reduce the content of collagen in MI scar.Grant Funding Source: Internal Award from the NYCOM Office of Research (E.I. Dedkov) and Research Grant from IRI Servier (R.J. Tomanek)
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
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.Conclusionour 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

AimWe 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.Methods105 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).ResultsPDCD5 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.ConclusionPDCD5 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.
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.)
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
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