Miracle-dz/UniFAR-FTUs huggingface dataset:
Proteolytic Release of the Intramolecular Chaperone Domain Confers Processivity to Endosialidase F*

Endosialidases (endoNs), as identified so far, are tailspike proteins of bacteriophages that specifically bind and degrade the α2,8-linked polysialic acid (polySia) capsules of their hosts. The crystal structure solved for the catalytic domain of endoN from coliphage K1F (endoNF) revealed a functional trimer. Folding of the catalytic trimer is mediated by an intramolecular C-terminal chaperone domain. Release of the chaperone from the folded protein confers kinetic stability to endoNF. In mutant c(S), the replacement of serine 911 by alanine prevents proteolysis and generates an enzyme that varies in activity from wild type. Using soluble polySia as substrate a 3-times higher activity was detected while evaluation with immobilized polySia revealed a 190-fold reduced activity. Importantly, activity of c(S) did not differ from wild type with tetrameric sialic acid, the minimal endoNF substrate. Furthermore, we show that the presence of the chaperone domain in c(S) destabilizes binding to polySia in a similar way as did selective disruption of a polySia binding site in the stalk domain. The improved catalytic efficiency toward soluble polySia observed in these mutants can be explained by higher dissociation and association probabilities, whereas inversely, an impaired processivity was found. The fact that endoNF is a processive enzyme introduces a new molecular basis to explain capsule degradation by bacteriophages, which until now has been regarded as a result of cooperative interaction of tailspike proteins. Moreover, knowing that release of the chaperone domain confers kinetic stability and processivity, conservation of the proteolytic process can be explained by its importance in phage evolution.
Clinical and histopathological aspects of the insect bite hypersensitivity in horses

In this study, the clinical and histopathological aspects of insect bite hypersensitivity (IBH) were assessed in horses. For this purpose, 12 horses with histopathologically confirmed cases of IBH of the skin were selected. The 12 animals were from 10 stud farms located in different municipalities of the state of Sao Paulo, Brazil. The main clinical findings included severe pruritus, alopecia and signs of irritability. The lesions were diffuse and predominantly dorsal, with the mane, tail and face most commonly affected. Histologically, perivascular lympho-histiocytic and eosinophilic dermatitis were also evident. Other findings included orthokeratotic hyperkeratosis, acanthosis and spongiosis. Thus, it was concluded that IBH could be diagnosed in horses through the combined use of clinical and epidemiological data and histopathological findings.
A Regulatory Role of the Bateman Domain of IMP Dehydrogenase in Adenylate Nucleotide Biosynthesis*

The Bateman domain (CBS subdomain) of IMP dehydrogenase (IMPDH), a rate-limiting enzyme of the de novo GMP biosynthesis, is evolutionarily conserved but has no established function. Deletion of the Bateman domain has no effect on the in vitro IMPDH activity. We report that in vivo deletion of the Bateman domain of IMPDH in Escherichia coli (guaBΔCBS) sensitizes the bacterium to growth arrest by adenosine and inosine. These nucleosides exert their growth inhibitory effect via a dramatic increase in the intracellular adenylate nucleotide pool, which results in the enhanced allosteric inhibition of PRPP synthetase and consequently a PRPP deficit. The ensuing starvation for pyrimidine nucleotides culminates in growth arrest. Thus, deletion of the Bateman domain of IMPDH derepresses the synthesis of AMP from IMP. The growth inhibitory effect of inosine can be rescued by second-site suppressor mutations in the genes responsible for the conversion of inosine to AMP (gsk, purA, and purB) as well as by the prsA1 allele, which encodes a PRPP synthetase that is insensitive to allosteric inhibition by adenylate nucleotides. Importantly, the guaBΔCBS phenotype can be complemented in trans by a mutant guaB allele, which encodes a catalytically disabled IMPDHC305A protein containing an intact Bateman domain. We conclude that the Bateman domain of IMPDH is a negative trans-regulator of adenylate nucleotide synthesis, and that this role is independent of the catalytic function of IMPDH in the de novo GMP biosynthesis.
Assessment of Effect of Leadership Behaviour Perceptions and Organizational Commitment of Hospital Employees on Job Satisfaction with Structural Equation Modelling

This article is dedicated to the memories of coal miners who lost their lives in Soma (Manisa). It was not their destiny. The purpose of this study was to establish a comprehensive causal model for leadership behaviour, organizational commitment and job satisfaction and to examine it with empirical data in the hospital employees. Structural equation model (SEM) was used to test the validity of constructs of leadership behaviour, organizational commitment and job satisfaction as well as to examine the causal relationship among them. The model was evaluated using data obtained from randomly selected 343 employees from Hacettepe University Adult Hospital in Ankara. The results showed that the measurement model for job satisfaction was a valid reliable model. In testing structural relationships, the results showed that leadership practices directly affect (r = 0.76) job satisfaction and indirectly affect (r = 0.095) it through organizational commitment. The results also showed that organizational commitment directly affects (r = 0.223) job satisfaction.
Phosphorylated Extracellular Signal-regulated Protein Kinases 1 and 2 Phosphorylate Sp1 on Serine 59 and Regulate Cellular Senescence via Transcription of p21Sdi1/Cip1/Waf1*

Expression of p21Sdi1 downstream of p53 is essential for induction of cellular senescence, although cancer cell senescence can also occur in the p53 null condition. We report herein that senescence-associated phosphorylated extracellular signal-regulated protein kinases 1 and 2 (SA-pErk1/2) enhanced p21Sdi1 transcription by phosphorylating Sp1 on Ser59 downstream of protein kinase C (PKC) α. Reactive oxygen species (ROS), which was increased in cellular senescence, significantly activated both PKCα and PKCβI. However, PKCα, but not PKCβI, regulated ROS generation and cell proliferation in senescent cells along with activation of cdk2, proven by siRNAs. PKCα-siRNA also reduced SA-pErk1/2 expression in old human diploid fibroblast cells, accompanied with changes of senescence phenotypes to young cell-like. Regulation of SA-pErk1/2 was also confirmed by using catalytically active PKCα and its DN-mutant construct. These findings strongly suggest a new pathway to regulate senescence phenotypes by ROS via Sp1 phosphorylation between PKCα and SA-pErk1/2: employing GST-Sp1 mutants and MEK inhibitor analyses, we found that SA-pErk1/2 regulated Sp1 phosphorylation on the Ser59 residue in vivo, but not threonine, in cellular senescence, which regulated transcription of p21Sdi1 expression. In summary, PKCα, which was activated in senescent cells by ROS strongly activated Erk1/2, and the SA-pErk1/2 in turn phosphorylated Sp1 on Ser59. Sp1-enhanced transcription of p21Sdi1 resulted in regulation of cellular senescence in primary human diploid fibroblast cells.
Metallothionein I as a direct link between therapeutic hematopoietic stem/progenitor cells and cerebral protection in stroke

Stroke continues to be a leading cause of death and disability worldwide, yet effective treatments are lacking. Previous studies have indicated that stem‐cell transplantation could be an effective treatment. However, little is known about the direct impact of transplanted cells on injured brain tissue. We wanted to help fill this knowledge gap and investigated effects of hematopoietic stem/progenitor cells (HSPCs) on the cerebral microcirculation after ischemia‐reperfusion injury (I/RI). Treatment of HSPCs in I/RI for up to 2 wk after cerebral I/RI led to decreased mortality rate, decreased infarct volume, improved functional outcome, reduced microglial activation, and reduced cerebral leukocyte adhesion. Confocal microscopy and fluorescence‐activated cell sorting analyses showed transplanted HSPCs emigrate preferentially into ischemic cortex brain parenchyma. We isolated migrated HSPCs from the brain; using RNA sequencing to investigate the transcriptome, we found metallothionein (MT, particularly MT‐I) transcripts were dramatically up‐regulated. Finally, to confirm the significance of MT, we exogenously administered MT‐I after cerebral I/RI and found that it produced neuroprotection in a manner similar to HSPC treatment. These findings provide novel evidence that the mechanism through which HSPCs promote repair after stroke maybe via direct action of HSPC‐derived MT‐I and could therefore be exploited as a useful therapeutic strategy for stroke. —Smith, H. K. , Omura, S. , Vital, S. A. , Becker, F. , Senchenkova, E. Y. , Kaur, G. , Tsunoda, I. , Peirce, S. M. , Gavins, F. N. E. Metallothionein I as a direct link between therapeutic hematopoietic stem/progenitor cells and cerebral protection in stroke. FASEB J. 32, 2381–2394 (2018). www.fasebj. org
Inhaled Drug Therapy in Chronic Obstructive Pulmonary Disease

The delivery of drugs to the lungs via the inhaled route has long been established as a successful and effective approach in the management of chronic obstructive pulmonary disease (COPD). Compared with oral administration, an inhaled drug directly targets the airways and allows a smaller drug dose to be used with the potential for a more rapid onset of action and less adverse systemic exposure. COPD is characterized by chronic airflow obstruction that is usually irreversible and progressive. Although most interventions do not affect disease progression, they can lead to significant improvements in quality of life. The mainstay of inhaled treatment revolves around relieving symptoms, optimizing lung function, improving exercise tolerance, and preventing exacerbations and long-term complications. A number of novel therapies are in clinical development for the treatment of patients with COPD and many of these agents have been shown to be toxic when given by parenteral administration. Targeted delivery of drug using the inhaled route to the local airways, or as a means to reach the systemic circulation, may allow an improved in vivo therapeutic profile for these novel treatments. The future development of inhaled drug therapies in the management of patients with COPD remains optimistic but challenging. This review focuses on the pharmacology and clinical application of inhaled drug therapies in the treatment of COPD and highlights evidence from the literature to support the rationale for
Loss of Specific Chaperones Involved in Membrane Glycoprotein Biosynthesis during the Maturation of Human Erythroid Progenitor Cells*

The production of erythrocytes requires the massive synthesis of red cell-specific proteins including hemoglobin, cytoskeletal proteins, as well as membrane glycoproteins glycophorin A (GPA) and anion exchanger 1 (AE1). We found that during the terminal differentiation of human CD34+ erythroid progenitor cells in culture, key components of the endoplasmic reticulum (ER) protein translocation (Sec61α), glycosylation (OST48), and protein folding machinery, chaperones BiP, calreticulin (CRT), and Hsp90 were maintained to allow efficient red cell glycoprotein biosynthesis. Unexpected was the loss of calnexin (CNX), an ER glycoprotein chaperone, and ERp57, a protein-disulfide isomerase, as well as a major decrease of the cytosolic chaperones, Hsc70 and Hsp70, components normally involved in membrane glycoprotein folding and quality control. AE1 can traffic to the cell surface in mouse embryonic fibroblasts completely deficient in CNX or CRT, whereas disruption of the CNX/CRT-glycoprotein interactions in human K562 cells using castanospermine did not affect the cell-surface levels of endogenous GPA or expressed AE1. These results demonstrate that CNX and ERp57 are not required for major glycoprotein biosynthesis during red cell development, in contrast to their role in glycoprotein folding and quality control in other cells.
Modulation of glutathione promotes apoptosis in triple‐negative breast cancer cells

Triple‐negative breast cancer has an extremely high rate of relapse. This is particularly due to the existence and survival of cancer stem cells (CSCs) characterized by increased amounts of glutathione (GSH). In this study, we evaluated the potential of pharmacological GSH depletion to sensitize CSCs to ionizing radiotherapy with an I‐125‐labeled nucleoside analog, 5‐iodo‐4'‐thio‐2'‐deoxyuridine (ITdU). CSCs were isolated using CD24‐‐ and CD44+‐specific microbeads. GSH and reactive oxygen species (ROS) were evaluated by fluorescence‐activated cell sorting. GSH synthesis was inhibited with buthionine sulfoximine (BSO). Apoptotic cells were identified with propidium iodide and double‐strand DNA breaks were detected by g‐H2AX staining. For therapy study, BSO treated and untreated mice xenografted with breast CSCs received weekly I‐125‐ITdU. Therapy efficiency was monitored by fluorodeoxyglucose‐18^‐positron emission tomography. We showed that GSH modulation sensitizes CD24‐ and CD44+ breast cancer cells to endogenous nanoradiotherapy. BSO synergistically affects ROS generation induced by I‐125‐ITdU. In an in vivo study, we demonstrated a complete tumor regression as a consequence of preconditioning with a GSH‐synthesis inhibitor prior to treatment with I‐125‐ITdU. GSH modulation in combination with an oxidative stress‐generating treatment such as endogenous radiotherapy using an Auger emitter offers an extraordinary opportunity for selective and efficient eradication of drug‐resistant CSCs. —Miran, T. , Vogg, A. T. J., Drude, N. , Mottaghy, F. M. , Morgenroth, A. Modulation of glutathione promotes apoptosis in triple‐negative breast cancer cells. FASEB J. 32,2803–2813 (2018). www.fasebj. org
Cell free hemoglobin in the fetoplacental circulation: a novel cause of fetal growth restriction?

Cell free hemoglobin impairs vascular function and blood flow in adult cardiovascular disease. In this study, we investigated the hypothesis that free fetal hemoglobin (fHbF) compromises vascular integrity and function in the fetoplacental circulation, contributing to the increased vascular resistance associated with fetal growth restriction (FGR). Women with normal and FGR pregnancies were recruited and their placentas collected freshly postpartum. FGR fetal capillaries showed evidence of erythrocyte vascular pacκing and extravasation. Fetal cord blood fHbF levels were higher in FGR than in normal pregnancies (P < 0.05) and the elevation of fHbF in relation to heme oxygenase‐1 suggests a failure of expected catabolic compensation, which occurs in adults. During ex vivo placental perfusion, pathophysiological fHbF concentrations significantly increased fetal‐side microcirculatory resistance (P < 0.05). fHbF sequestered NO in acute and chronic exposure models (P <0.001), and fHbF‐primed placental endothelial cells developed a proinflammatory phenotype, demonstrated by activation of NF‐κB pathway, generation of IL‐1ä and TNF‐ä (both P < 0.05), uncontrolled angiogenesis, and disruption of endothelial cell flow alignment. Elevated fHbF contributes to increased fetoplacental vascular resistance and impaired endothelial protection. This unrecognized mechanism for fetal compromise offers a novel insight into FGR as well as a potential explanation for associated poor fetal outcomes such as fetal demise and stillbirth. —Brooκ, A. , Hoaκsey, A. , Gurung, R. , Yoong, E. E. C. , Sneyd, R. , Baynes, G. C. , Bischof, H. , Jones, S. , Higgins, L. E. , Jones, C. , Greenwood, S. L. , Jones, R. L. , Gram, M. , Lang, I. , Desoye, G. , Myers, J., Schneider, H. , Hansson, S. R. , Crocκer, I. P., Brownbill, P. Cell free hemoglobin in the fetoplacental circulation: a novel cause of fetal growth restriction? FASEB J. 32, 5436–5446 (2018). www.fasebj. org
Comparing Pooled Peptides with Intact Protein for Accessing Cross-presentation Pathways for Protective CD8+ and CD4+ T Cells*

To better understand the mechanisms of intracellular trafficking and presentation of exogenous peptides by antigen-presenting cells (APC), we compared the handling of overlapping 24-mer peptides from HIV Nef either mixed or covalently linked in tandem in one protein. Once internalized, peptides trafficked not only to endosomes but also to cytosol, and activated CD8+ and CD4+ T cells. In contrast, whole protein was found to traffic only to the endosomal compartments, and primarily activated CD4+ T cells. Finally, with adjuvant, overlapping peptides were capable of protecting against lethal viral challenge, whereas the intact protein was less protective. These data suggest that overlapping long peptides are cross-presented through more varied intracellular routes and are more efficient in priming protective immunity than the whole protein.
Interdomain Flexibility in Full-length Matrix Metalloproteinase-1 (MMP-1)*

The presence of extensive reciprocal conformational freedom between the catalytic and the hemopexin-like domains of full-length matrix metalloproteinase-1 (MMP-1) is demonstrated by NMR and small angle x-ray scattering experiments. This finding is discussed in relation to the essentiality of the hemopexin-like domain for the collagenolytic activity of MMP-1. The conformational freedom experienced by the present system, having the shortest linker between the two domains, when compared with similar findings on MMP-12 and MMP-9 having longer and the longest linker within the family, respectively, suggests this type of conformational freedom to be a general property of all MMPs.
Determination of Ethylene Glycol Tissue Content After Fatal Oral Poisoning and Pathologic Findings

A 23-year-old comatose man who had drunk an unknown amount of ethylene glycol was admitted to the hospital 5 hours after ingestion. The initial plasma ethylene glycol concentration was 116.2 mg/100 ml. A severe metabolic acidosis was present. Despite aggressive therapy with ethanol, hemodialysis, and intensive care support, the patient died 27 hours after poisoning. The plasma ethylene glycol concentration immediately before death was 35.9 mg/100 ml. Brain edema and acute renal tubular necrosis were evident at postmortem examination. Oxalate crystals were identified in both organs. Ethylene glycol content or concentration was determined in tissues and biologic fluids.
Epithelial and Mesenchymal Cell Biology The Aryl Hydrocarbon Receptor Ligand ITE Inhibits TGF 1-Induced Human Myofibroblast Differentiation

Fibrosis can occur in any human tissue when the normal wound healing response is amplified. Such amplification results in fibroblast proliferation, myofibroblast differentiation, and excessive extracellular matrix deposition. Occurrence of these sequelae in organs such as the eye or lung can result in severe consequences to health. Unfortunately, medical treatment of fibrosis is limited by a lack of safe and effective therapies. These therapies may be developed by identifying agents that inhibit critical steps in fibrotic progression; one such step is myofibroblast differentiation triggered by transforming growth factor1 (TGF 1). In this study, we demonstrate that TGF 1induced myofibroblast differentiation is blocked in human fibroblasts by a candidate endogenous aryl hydrocarbon receptor (AhR) ligand 2-(1=H-indole3=-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE). Our data show that ITE disrupts TGF 1 signaling by inhibiting the nuclear translocation of Smad2/3/4. Although ITE functions as an AhR agonist, and biologically persistent AhR agonists, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, cause severe toxic effects, ITE exhibits no toxicity. Interestingly, ITE effectively inhibits TGF 1-driven myofibroblast differentiation in AhR / fibroblasts: Its ability to inhibit TGF 1 signaling is AhR independent. As supported by the results of this study, the small molecule ITE inhibits myofibroblast differentiation and may be useful clinically as an antiscarring agent. (Am J Pathol 2011, 178:1556–1567; DOI: 10.1016/j.ajpath.2010.12.025)
Esmolol-Induced Regional Wall Motion Abnormalities Do Not Affect Regional Ventricular Elastances

The effect of regional wall motion abnormalities (RWMA) on regional and global left ventricular (LV) elastances has not been defined. To induce RWMA, we infused esmolol (9 mg) into the left anterior descending coronary artery in eight anesthetized open-chest canine preparations. Global and regional stroke volumes and end-systolic pressure-volume relationships (LV conductance catheter) and pressure-length relationships (sonomicrometer crystals) were measured in dysfunctional (apical) and normal (basilar) LV regions at baseline, during esmolol infusion, and after treatment with a systemic dobutamine infusion (4 &mgr;g · kg−1 · min−1) and combined dobutamine-esmolol. Esmolol induced apical dyskinesis, as evidenced by reduced effective apical stroke volumes and stroke work, a parallel right-shift of the apical regional, global end-systolic pressure-volume relationships, and increased regional and global LV volumes (P < 0.05). However, global and regional elastances remained unchanged. Dobutamine increased global and apical regional elastances, but did not increase regional volumes. During the infusion of combined esmolol-dobutamine, apical elastance and volumes increased compared with baseline (P < 0.05), but apical regional stroke work decreased. Thus, esmolol-induced RWMA were associated with cardiac dilation but not with decreased regional or global elastances. Implications Regional and global elastances and maximal stroke volumes may not identify esmolol-induced left ventricular regional dysfunction in dogs. The primary effect of asynchrony of regional contraction is global cardiac dilation. Systemic dobutamine infusion increases regional and global left ventricular elastances but does not reverse regional wall motion abnormality-induced cardiac dilation.
Epithelial and Mesenchymal Cell Biology Type 2 Helper T-Cell Cytokines Induce Morphologic and Molecular Characteristics of Atopic Dermatitis in Human Skin Equivalent

Both the immune system and the epidermis likely have an important role in the pathogenesis of atopic dermatitis (AD). The objective of the present study was to develop a human skin equivalent model exhibiting morphologic and molecular characteristics of AD in a controlled manner. Skin equivalents generated from normal adult human keratinocytes were stimulated with type 2 T-helper cell (Th2) cytokines IL-4 and IL-13, and morphologic features and gene expression of the epidermis were studied. Th2 cytokines induced intercellular edema similar to spongiotic changes observed in lesional AD as assessed at histopathologic analysis and electron microscopy. Furthermore, genes known to be specifically expressed in epidermis of patients with AD such as CAII and NELL2 were induced. In contrast, expression of psoriasis-associated genes such as elafin and hBD2 was not changed. Th2 cytokines caused DNA fragmentation in the keratinocytes, which could be inhibited by the caspase inhibitor Z-VAD, which suggests that apoptosis was induced. In addition, up-regulation of the death receptor Fas was observed in keratinocytes after Th2 cytokine stimulation. IL-4 and IL-13 induced phosphorylation of the signaling molecule STAT6. It was concluded that the skin equivalent model described herein may be useful in investigation of the epidermal aspects of AD and for study of drugs that act at the level of keratinocyte biology. (Am J Pathol 2011, 178:2091–2099; DOI: 10.1016/j.ajpath.2011.01.037)