gebhart/patrepeval huggingface dataset:
Sustained Activation of a G Protein-coupled Receptor via “Anchored” Agonist Binding (1996)

An inherent therapeutic limitation of many G protein-coupled receptor agonists is a short duration of action due to rapid dissociation from receptors. Salmeterol is a modified beta-adrenergic receptor (betaAR) agonist that has a long duration of action at the beta2AR (but not the beta1AR) both in vitro and in vivo and that is persistent despite extensive washout of the agonist. It has been proposed that salmeterol binds not only to the active site of the beta2AR (localized to receptor transmembrane spanning domains (TMDs) 3 and 5) but also to another site (termed the "exosite") that anchors it to the receptor and provides for repetitive active-site binding events. To identify the location of this exosite, we used site-directed mutagenesis to replace beta2AR amino acids 149-173 (within TMD4) with beta1AR sequence. The resulting constructs were then expressed in COS-7 cells for radioligand binding studies. Using this approach, when this domain was replaced with the analogous beta1AR sequence, the ability of salmeterol to persist at the receptor under washout conditions was reduced by 67%. The results from more selective mutants (S-(149-166), S-(164-173), and S-(149-158)) indicated that a limited 10-amino acid region (beta2AR residues 149-158), localized at the interface of the cytoplasm and the transmembrane domain, contains a critical determinant for exosite binding. Whereas CHW cells stably expressing wild-type beta2AR displayed persistent salmeterol-promoted cAMP accumulation despite agonist washout, substitution of beta2AR residues 149-158 with beta1AR sequence resulted in a 56% attenuation of salmeterol-promoted cAMP accumulation under identical washout conditions. A reverse chimera was also studied, which consisted of a substitution of beta2AR residues 152-156 into the beta1AR. This substitution was found to confer exosite binding to the beta1AR. None of these mutations decreased the affinity of salmeterol for the receptor at the active site as assessed in competition binding studies. Anchored binding to this motif thus represents a novel mechanism by which agonists like salmeterol can repetitively activate receptors. Conceivably, with other G protein-coupled receptors that have similar motifs, anchored ligands can be designed to provide for long durations of action by this mechanism.
Rabbit and rat platelets do not respond to thrombin receptor peptides that activate human platelets (1993)

Human platelets are aggregated and induced to release their granule contents and form thromboxane by peptides as short as 6-amino acid residues (SFLLRN) corresponding to the newly released N-terminus of the thrombin receptor that is cleaved by thrombin. Using washed platelets, we found that these responses to SFLLRN (2 to 6 mumol/L) were enhanced by fibrinogen. However, neither SFLLRN nor SFLLRNPNDKYEPF had any effect on washed rabbit or rat platelets, although they were fully responsive to human thrombin. Concentrations of the peptides as high as 100 mumol/L did not cause the platelets of rabbits or rats to change shape, aggregate, release granule contents, or form thromboxane. SFLLRN did not affect the extent of aggregation induced by adenosine diphosphate (ADP) or a low concentration of thrombin. Pig platelets responded to 50 mumol/L SFLLRN with reversible aggregation, which was enhanced by fibrinogen, but not accompanied by the release of dense granule contents. Guinea pig platelets aggregated and released granule contents in response to 25 or 50 mumol/L of SFLLRN, but responded with only shape change to lower concentrations. Thus, these experiments indicate that rabbit and rat platelets lack a functional response to human thrombin receptor peptides that fully activate the previously described human thrombin receptor, despite a full response of both rabbit and rat platelets to human thrombin, and that pig and guinea pig platelets have incomplete responses to these human thrombin receptor peptides. The results suggest that platelets of rabbits and rats, and perhaps guinea pigs and pigs, respond to thrombin through an alternative receptor that has also been suggested to be present on human platelets.
A Bronchial Genomic Classifier for the Diagnostic Evaluation of Lung Cancer (2015)

The gene-expression classifier improved the diagnostic performance of bronchoscopy for the detection of lung cancer. In intermediate-risk patients with a nondiagnostic bronchoscopic examination, a negative classifier score provides support for a more conservative diagnostic approach. (Funded by Allegro Diagnostics and others; AEGIS-1 and AEGIS-2 ClinicalTrials.gov numbers, NCT01309087 and NCT00746759.).
On the Origin of the Thermostabilization of Proteins Induced by Sodium Phosphate (2005)

The B1 domain of protein L shows a linear rise in thermostability with increasing concentrations of sodium phosphate. Equal behavior is observed for a set of mutant proteins where surface lysines are mutated to noncharged residues, but the mutant's thermostabilities show different sensitivities to phosphate, encoded in the varying slopes observed (mi). The melting temperature in the absence of the cosolute also correlates linearly with mi. The stabilizing effect of the phosphate ion reaches a saturation point, which has been experimentally determined for protein L (1610 mM phosphate). These results indicate that the phosphate-induced stabilization is an inherent property of the protein, encoded in the amino acid sequence. Changes in stability upon mutation are attributed to a redistribution of the overall network of solvated surface charges. Stabilization by phosphate is understood in terms of interactions with the protein surface, reducing the unfavorable contacts between like charges, maximizing the number of accessible conformations of the surface-charged side chains, and optimizing solvation.
Chronic antidepressant administration increases the expression of cAMP response element binding protein (CREB) in rat hippocampus (1996)

The present study demonstrates that chronic, but not acute, adminstration of several different classes of antidepressants, including serotonin- and norepinephrine-selective reuptake inhibitors, increases the expression of cAMP response element binding protein (CREB) mRNA in rat hippocampus. In contrast, chronic administration of several nonantidepressant psychotropic drugs did not influence expression of CREB mRNA, demonstrating the pharmacological specificity of this effect. In situ hybridization analysis demonstrates that antidepressant administration increases expression of CREB mRNA in CA1 and CA3 pyramidal and dentate gyrus granule cell layers of the hippocampus. In addition, levels of CRE immunoreactivity and of CRE binding activity were increased by chronic antidepressant administration, which indicates that expression and function of CREB protein are increased along with its mRNA. Chronic administration of the phosphodiesterase (PDE) inhibitors rolipram or papaverine also increased expression of CREB mRNA in hippocampus, demonstrating a role for the cAMP cascade. Moreover, coadministration of rolipram with imipramine resulted in a more rapid induction of CREB than with either treatment alone. Increased expression and function of CREB suggest that specific target genes may be regulated by these treatments. We have found that levels of brain-derived neurotrophic factor (BDNF) and trkB mRNA are also increased by administration of antidepressants or PDE inhibitors. These findings indicate that upregulation of CREB is a common action of chronic antidepressant treatments that may lead to regulation of specific target genes, such as BDNF and trkB, and to the long-term effects of these treatments on brain function.
Role of JNK Translocation to Mitochondria Leading to Inhibition of Mitochondria Bioenergetics in Acetaminophen-induced Liver Injury (2008)

Previously, we demonstrated JNK plays a central role in acetaminophen (APAP)-induced liver injury (Gunawan, B. K., Liu, Z. X., Han, D., Hanawa, N., Gaarde, W. A., and Kaplowitz, N. (2006) Gastroenterology 131, 165-178). In this study, we examine the mechanism involved in activating JNK and explore the downstream targets of JNK important in promoting APAP-induced liver injury in vivo. JNK inhibitor (SP600125) was observed to significantly protect against APAP-induced liver injury. Increased mitochondria-derived reactive oxygen species were implicated in APAP-induced JNK activation based on the following: 1) mitochondrial GSH depletion (maximal at 2 h) caused increased H2O2 release from mitochondria, which preceded JNK activation (maximal at 4 h); 2) treatment of isolated hepatocytes with H2O2 or inhibitors (e.g. antimycin) that cause increased H2O2 release from mitochondria-activated JNK. An important downstream target of JNK following activation was mitochondria based on the following: 1) JNK translocated to mitochondria following activation; 2) JNK inhibitor treatment partially protected against a decline in mitochondria respiration caused by APAP treatment; and 3) addition of purified active JNK to mitochondria isolated from mice treated with APAP plus JNK inhibitor (mitochondria with severe GSH depletion, covalent binding) directly inhibited respiration. Cyclosporin A blocked the inhibitory effect of JNK on mitochondria respiration, suggesting JNK was directly inducing mitochondrial permeability transition in isolated mitochondria from mice treated with APAP plus JNK inhibitor. Addition of JNK to mitochondria isolated from control mice did not affect respiration. Our results suggests that APAP-induced liver injury involves JNK activation, due to increased reactive oxygen species generated by GSH-depleted mitochondria, and translocation of activated JNK to mitochondria where JNK induces mitochondrial permeability transition and inhibits mitochondria bioenergetics.
Herpes Simplex Virus 1 Enters Human Keratinocytes by a Nectin-1-Dependent, Rapid Plasma Membrane Fusion Pathway That Functions at Low Temperature (2016)

The gold standard of antiviral treatment for any human virus infection is the prevention of virus entry into the host cell. In the case of HSV-1, primary infection in the human begins in the epidermis of the skin or the oral mucosa, where the virus infects keratinocytes, and it is therefore important to understand the molecular events involved in HSV-1 entry into this cell type. Nonetheless, few studies have looked specifically at entry into these relevant human cells. Our results reveal a new route for virus entry that is specific to keratinocytes, involves rapid entry, and functions at low temperatures. This may reflect the environmental conditions encountered by HSV-1 when entering its host through the skin and emphasizes the importance of studying virus-host interactions in physiologically relevant cells.
Cross Protection between an Encapsulated Strain of Staphylococcus hyicus and an Encapsulated Strain of Staphylococcus epidermidis (1990)

Strain ST67P of Staphylococcus hyicus was capsular type I (++)/II(+) of S. epidermidis as determined by the method of Ichiman. To mice immunized with heat-killed vaccine of strain ST67P, homologous strain and strain ATCC 31432 (capsular type I), SE-360 (capsular type II) and SE-10 (capsular type III) of S. epidermidis were injected intraperitoneally into mice, then, the viable cell number of the organisms in the peritoneal cavity were enumerated of 30 minutes and 20 hours after the injection. Results showed that the viable cell number of the homologous strain and strain ATCC 31432 was remarkably decreased at 20 hours after the injection, however, the cells were increased with strain SE-360 and SE-10. Passive protective activity of rabbit anti-ATCC 31432 serum was absorbed either with homologous strain or strain ST67P in the mouse, however, protective activity of anti-SE-360 strain serum and anti-SE-10 strain serum was not absorbed with these organisms although the activity was absorbed with homologous organisms. With an ultrathin section preparation of strain ST67P conjugated with ferritin-labelled rabbit anti-homologous strain serum, numerous ferritin granules surrounding the outermost layer of large capsule were electronmicro-scopically demonstrated. In the same organisms treated with ferritin-labelled anti-ATCC31432 strain serum, the all walls were surrounded by a relatively thinner capsule and a number of ferritin granules were located in the outermost layer of the capsule. However, with the organisms treated with ferritin-labelled anti-SE-360 strain serum only a number of ferritin granules were shown on the surface of the cell walls, and neither capsule nor ferritin granules were exhibited in the organisms treated with ferritin-labelled anti-SE-10 strain serum.
Splanchnic and Renal Sympathetic Activity in Relation to Hemodynamics During Isoflurane Administration in Pigs (1995)

The aim of the present study was to investigate the impact of isoflurane on regional neurogenic mechanisms in the control of vascular tone. Therefore, regional determinations of sympathetic activity and hemodynamics were made in chloralose-anesthetized swine before and during administration of 1.4% isoflurane. Sympathetic activity was examined from spillover of norepinephrine (NE) into the circulation using an isotope dilution technique. Administration of isoflurane caused a marked decrease in mesenteric (65 +/- 9 pmol.min-1.100 g-1; P < 0.05) NE spillover. Renal NE spillover was moderately decreased (25 +/- 6 pmol.min-1.100 g-1; P < 0.05), whereas liver NE spillover did not change significantly during isoflurane administration, suggesting that liver sympathetic activity is maintained at this level of isoflurane anesthesia. Total body NE spillover decreased (13 +/- 2 pmol.min-1.100 g-1, P < 0.05). Thus, isoflurane affected sympathetic outflow in a regionally differentiated pattern. Significant correlations were found between total body, mesenteric, and renal NE spillovers and vascular resistances, supporting the concept that the observed reductions in vascular resistances in these circulations during isoflurane administration were in part a consequence of reduced sympathetic outflow. In the liver circulation, no correlation was found between NE spillover and liver portal or liver arterial vascular resistances. Liver arterial resistance was significantly reduced during isoflurane administration while liver portal resistance was unchanged. Administration of isoflurane caused reductions in cardiac output, renal, portal, hepatic arterial, and total hepatic blood flows, whereas mesenteric blood flow was unchanged. To summarize, isoflurane decreased mesenteric and renal NE spillover with concomitant reductions in vascular resistances.(ABSTRACT TRUNCATED AT 250 WORDS)
Site-specific recombinases: changing partners and doing the twist (1986)

Thirty-five years have passed since the discovery of lysogenic induction of bacteriophage ( 46), and over the years it has become evident that site-specific integration of the phage chromosome into the bacterial genome is a key element of this process.While much of the genetic and biochemical data were derived from the phage lambda paradigm (73), site-specific recombination is not merely a curiosity confined to an arcane organism like lambda, but an important process controlling the development of many organisms as diverse as temperate bacteriophages and hu- mans.The list of functions performed by site-specific recom- bination systems has grown to include, in addition to the integration and excision of bacteriophage chromosomes (15, 73), resolution of cointegrate structures generated during the "hopping" of transposons like Tn3 (7,60,61), switching of flagellar antigens promoted by the Hin protein of Salmonella typhimurium (66), control of the host range of phages like Mu by the Gin gene product (42, 69), maintenance of faithful segregation of lysogenic phage P1 by the Cre protein (8), expression of nitrogen fixation genes during heterocyst differentiation in cyanobacteria (25), the possible maintenance of high copy number of the 2pum plasmid of Saccharomyces cerevisiae by the plasmid-encoded FLP gene product (B.Futcher, personal communication), and generation of a diversity of antibodies (11, 22) and T-cell receptors of the immune system (16,23,33).In hope of discovering universal biochemical mechanisms underlying these important processes, several groups have followed the lead of Nash and colleagues (53) and have developed systems that reproduce site-specific recombination in vitro.In this review I shall examine five such systems and point out similarities and differences among them.In addition to the A integrase system, these systems include the resolvases of transposons Tn3 and yb (60), the Cre protein of phage P1 (5), the Gin protein of phage Mu (47, 58), the Hin protein of the phase inversion system of S. typhimurium (invertases) (38), and the FLP protein of the 2,um plasmid of S. cerevisiae (48,70).Common features of site-specific recombinases.In his original formulation, Campbell (15) postulated that integration of the lambda genome occurred by reciprocal recombination between regions of homology on the circular phage chromo- some and that of Escherichia coli.This region of exact homology is now known to be small, only 15 base pairs (bp), and crossing over occurs between the phage attachment site (attP [-240 bp long]) and its bacterial equivalent (attB [-25 bp long]) (see Fig. 1 and2).The hallmark of all site-specific recombinases is that they promote reciprocal recombination t This article is dedicated to Lou Siminovitch on the occasion of his 65th birthday.
Abstract 5011: Cancer immunotherapy targeting CD47: Wild type SIRPαFc is the ideal CD47-blocking agent to minimize unwanted erythrocyte binding (2014)

Abstract Background: CD47 binds to SIRPα on the surface of macrophages and delivers a “do not eat” signal that suppresses phagocytosis. There is strong evidence that many hematopoietic and solid tumors exploit the CD47-SIRPα pathway to escape macrophage-mediated destruction, and blocking CD47 has emerged as a promising cancer immunotherapy. There are several approaches available to achieve CD47 blockade, including wild type SIRPαFc fusion proteins, engineered high affinity SIRPαFcs and monoclonal antibodies. One concern with CD47-based therapies is the expression of the target on the surface of red blood cells (RBCs), which has the potential to act as a large antigen sink and cause hematological toxicity. Indeed, anemia has been reported in animals treated with high affinity SIRPαFcs variants and CD47-specific antibodies. Methods: Various CD47-specific biologics, including SIRPαFc fusion proteins containing wild type or mutant SIRPα sequences and CD47-specific antibodies were evaluated for binding to erythrocytes and other cell types by flow cytometry. Results: SIRPαFc fusion proteins containing wild type SIRPα sequences, which bind to CD47 with nanomolar affinity, bound very poorly to human RBCs compared to both blocking and non-blocking CD47 antibodies. This striking difference was highly reproducible and occurred across different blood types, but was not seen with other cells (e.g., tumor cell lines), indicating an erythrocyte-specific phenomenon. The presence of wild type SIRPα sequences was critical to achieving a low RBC binding profile, as mutated SIRPαFc with enhanced CD47 affinity bound strongly to human erythrocytes. Interestingly, wild type SIRPαFc binding to human RBCs was dramatically increased when CD47 was first clustered with a non-blocking antibody, suggesting that wild type SIRPαFc normally lacks the ability to aggregate CD47 on the erythrocyte surface. Finally, the lack of appreciable SIRPαFc binding to erythrocytes is unique to humans, as non-mutated mouse SIRPαFc could bind mouse RBCs and wild type human SIRPαFc cross reacts with CD47 on the surface of non-human primate and porcine RBCs. Conclusions: Wild type SIRPαFc, unlike other CD47 blocking agents, exhibits very low binding to human erythrocytes. This predicts that wild type SIRPαFc will have superior pharmacokinetic properties and less toxicity in cancer patients. Furthermore, the strong binding of wild type SIRPαFc to monkey RBCs suggests that preclinical studies in non-human primates may overestimate the risk of hematological toxicity in humans. Citation Format: Robert A. Uger, Karen Dodge, Xinli Pang, Penka S. Petrova. Cancer immunotherapy targeting CD47: Wild type SIRPαFc is the ideal CD47-blocking agent to minimize unwanted erythrocyte binding. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5011. doi:10.1158/1538-7445.AM2014-5011
Myocardial Infarction and Cardiac Remodelling in Mice (2002)

We established a mouse model of cardiac dysfunction due to myocardial infarction (MI). For this we ligated the left anterior descending coronary artery in male C57BL/6J mice and assessed healing and left ventricular (LV) remodelling at 1, 2 and 4 days and 1, 2 and 4 weeks after MI. Echocardiography was performed at 1 and 2 weeks and 1, 2, 4 and 6 months after MI. We found that neutrophil infiltration of the infarct border was noticeable at 1-2 days. Marked macrophage infiltration occurred at day 4, while lymphocyte infiltration was apparent at 7-14 days. Massive proliferation of fibroblasts and collagen accumulation began by day 7-14, and scar formation was completed by day 21. LV diastolic dimension increased markedly at 2 weeks and remained at the same level thereafter. LV shortening fraction decreased significantly at 2 weeks and then slowly decreased. In non-infarcted areas of the LV, myocyte cross-sectional area and interstitial collagen fraction increased progressively, reaching a maximum at 4 months. This study provides important qualitative and quantitative information about the natural history of cardiac remodelling after MI in mice.
Autotransplantation of Pancreatic Fragments to the Portal Vein and Spleen of Totally Pancreatectomized Dogs (1978)

Forty-nine dogs were made diabetic by total pancreatectomy. Fifteen untreated pancreatectomized animals survived a mean (+/-S.E.) of 7.0 +/- 1.1 days with a mean (+/-S.E.) plasma glucose level of 402 +/- 26 mg/100 ml before death. The pancreata of 32 dogs were distended with cold (4 degrees ) Hanks' solution, minced, digested with collagenase (600 U/ml tissue) for 15-25 minutes, and autotransplanted either into the splenic artery (three dogs), directly into the splenic pulp (21 dogs), or into the portal vein (ten dogs). Tissue infusion into the splenic artery resulted in infarction and persistent hyperglycemia. Direct implantation into the splenic pulp of tissue digested for 15, 20 and 25 minutes resulted in permanent normoglycemia (fasting plasma glucose < 150 mg/100 ml) in 7 of 8, 7 of 7, and 6 of 6 dogs respectively. Glucose tolerance test mean (+/-S.E.) K values (% decline of plasma glucose concentration/minute) in these groups two weeks after transplantation were 1.20 +/- 0.20%, 1.60 +/- 0.25 and 0.70 0.08% respectively, indicating that 20 minutes digestion was best for intrasplenic transplantation. Tissue prepared in the optimal manner (20 minutes digestion) and embolized into the liver resulted in normoglycemia in three of eight dogs, and a mean (+/-S.E.) K value of 0.77 +/- 0.10%. Both dogs receiving tissue dispersed for 25 minutes into the portal vein remained hyperglycemic. In the dogs subjected to intraportal transplantation, portal pressure rose from a mean (+/-S.E.) of 6.5 +/- 0.6 cm H(2)O before to 21.9 +/- 2.2 cm H(2)O immediately after tissue embolization, but declined to 6.5 +/- 1.0 cm H(2)O by ten weeks in animals becoming normoglycemic. We conclude that in dogs direct implantation of pancreatic tissue into the splenic pulp is superior to embolization into the portal vein or splenic artery because the splenic circulation is not compromized, portal hypertension is obviated, and glucose metabolism is best controlled as judged by glucose tolerance test K values.