scholarly journals Characterization of a potent human interleukin-11 agonist

2003 ◽  
Vol 375 (1) ◽  
pp. 23-32 ◽  
Author(s):  
Dimitri HARMEGNIES ◽  
Xiao-Ming WANG ◽  
Paul VANDENBUSSCHE ◽  
Arnaud LEON ◽  
Patricia VUSIO ◽  
...  
Keyword(s):  
Biological Activities ◽  
Gel Filtration ◽  
Electrophoretic Analysis ◽  
Receptor Complex ◽  
Wild Type ◽  
Α Subunit ◽  
Receptor Chain ◽  
Interleukin 11 ◽  
New Protein

Human interleukin-11 (hIL-11) is a multi-potential cytokine that is involved in numerous biological activities, such as haematopoiesis, osteoclastogenesis, neurogenesis and female fertility, and also displays anti-inflammatory properties. IL-11 is used clinically to treat chemotherapy-induced thrombocytopenia. Because of its broad spectrum of action, improved IL-11 agonists, as well as IL-11 antagonists, could be of interest for numerous clinical applications. IL-11 signalling is dependent on the formation of a tripartite ligand–receptor complex consisting of IL-11, the IL-11R (IL-11 receptor) α subunit (responsible for the specificity of the interaction) and gp130 (glycoprotein 130) receptor β subunit (responsible for signal transduction). The interaction between IL-11 and IL-11Rα subunit occurs at its recently assigned site I. We have designed an IL-11 mutein whose hydrophobicity at site I has been increased. The mutein has been characterized in terms of structure, affinity, specificity and bioactivity. Electrophoretic analysis, gel filtration, IR spectroscopy and CD indicate that this new protein is more compact than wild-type IL-11. It binds to IL-11Rα with a three-fold-enhanced affinity, and retains the ability to recruit gp130 through site II. However, analysis of its biological activity revealed a complex pattern: although this mutein is 60–400-fold more active than wild-type IL-11 on the proliferation of 7TD1 murine hybridoma cell, it is less active than IL-11 on the proliferation of B9 cells, another murine hybridoma cell line. The results are interpreted on the basis of an IL-11 conformational change induced by the mutations, and the preferential use by the mutein of another unknown transducing receptor chain.

scholarly journals Identification and Characterization of the Phage Gene sav, Involved in Sensitivity to the Lactococcal Abortive Infection Mechanism AbiV

2009 ◽  
Vol 75 (8) ◽  
pp. 2484-2494 ◽  
Author(s):  
Jakob Haaber ◽  
Geneviève M. Rousseau ◽  
Karin Hammer ◽  
Sylvain Moineau
Keyword(s):  
Gel Filtration ◽  
Point Mutations ◽  
Whole Genome ◽  
Wild Type ◽  
Abortive Infection ◽  
Native Form ◽  
Phage Gene ◽  
Infection Mechanism ◽  
Identification And Characterization

ABSTRACT Lactococcus lactis phage mutants that are insensitive to the recently characterized abortive infection mechanism AbiV were isolated and analyzed in an effort to elucidate factors involved in the sensitivity to AbiV. Whole-genome sequencing of the phage mutants p2.1 and p2.2 revealed mutations in an orf that is transcribed early, indicating that this orf was responsible for AbiV sensitivity. Sequencing of the homologous regions in the genomes of other AbiV-insensitive mutants derived from p2 and six other lactococcal wild-type phages revealed point mutations in the homologous orf sequences. The orf was named sav (for sensitivity to AbiV), and the encoded polypeptide was named SaV. The purification of a His-tagged SaV polypeptide by gel filtration suggested that the polypeptide formed a dimer in its native form. The overexpression of SaV in L. lactis and Escherichia coli led to a rapid toxic effect. Conserved, evolutionarily related regions in SaV polypeptides of different phage groups are likely to be responsible for the AbiV-sensitive phenotype and the toxicity.

scholarly journals Dysfunctional proofreading in the Escherichia coli DNA polymerase III core

2004 ◽  
Vol 384 (2) ◽  
pp. 337-348 ◽  
Author(s):  
Duane A. LEHTINEN ◽  
Fred W. PERRINO
Keyword(s):  
Dna Polymerase ◽  
Gel Filtration ◽  
Exonuclease Activity ◽  
Dna Polymerase Iii ◽  
Wild Type ◽  
Α Subunit ◽  
Polymerase Iii ◽  
Pol Iii

The ε-subunit contains the catalytic site for the 3′→5′ proofreading exonuclease that functions in the DNA pol III (DNA polymerase III) core to edit nucleotides misinserted by the α-subunit DNA pol. A novel mutagenesis strategy was used to identify 23 dnaQ alleles that exhibit a mutator phenotype in vivo. Fourteen of the ε mutants were purified, and these proteins exhibited 3′→5′ exonuclease activities that ranged from 32% to 155% of the activity exhibited by the wild-type ε protein, in contrast with the 2% activity exhibited by purified MutD5 protein. DNA pol III core enzymes constituted with 11 of the 14 ε mutants exhibited an increased error rate during in vitro DNA synthesis using a forward mutation assay. Interactions of the purified ε mutants with the α- and θ-subunits were examined by gel filtration chromatography and exonuclease stimulation assays, and by measuring polymerase/exonuclease ratios to identify the catalytically active ε511 (I170T/V215A) mutant with dysfunctional proofreading in the DNA pol III core. The ε511 mutant associated tightly with the α-subunit, but the exonuclease activity of ε511 was not stimulated in the α–ε511 complex. Addition of the θ-subunit to generate the α–ε511–θ DNA pol III core partially restored stimulation of the ε511 exonuclease, indicating a role for the θ-subunit in co-ordinating the α–ε polymerase–exonuclease interaction. The α–ε511–θ DNA pol III core exhibited a 3.5-fold higher polymerase/exonuclease ratio relative to the wild-type DNA pol III core, further indicating dysfunctional proofreading in the α–ε511–θ complex. Thus the ε511 mutant has wild-type 3′→5′ exonuclease activity and associates physically with the α- and θ-subunits to generate a proofreading-defective DNA pol III enzyme.

scholarly journals Identification of Redeye, a new sleep-regulating protein whose expression is modulated by sleep amount

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Mi Shi ◽  
Zhifeng Yue ◽  
Alexandre Kuryatov ◽  
Jon M Lindstrom ◽  
Amita Sehgal
Keyword(s):  
Homeostatic Regulation ◽  
Wild Type ◽  
Α Subunit ◽  
Forward Genetic Screen ◽  
Protein Levels ◽  
Severe Reduction ◽  
Sleep Homeostat ◽  
Sleep Length ◽  
Sleep Amount ◽  
New Protein

In this study, we report a new protein involved in the homeostatic regulation of sleep in Drosophila. We conducted a forward genetic screen of chemically mutagenized flies to identify short-sleeping mutants and found one, redeye (rye) that shows a severe reduction of sleep length. Cloning of rye reveals that it encodes a nicotinic acetylcholine receptor α subunit required for Drosophila sleep. Levels of RYE oscillate in light–dark cycles and peak at times of daily sleep. Cycling of RYE is independent of a functional circadian clock, but rather depends upon the sleep homeostat, as protein levels are up-regulated in short-sleeping mutants and also in wild type animals following sleep deprivation. We propose that the homeostatic drive to sleep increases levels of RYE, which responds to this drive by promoting sleep.

scholarly journals Human interferon-ϵ and interferon-κ exhibit low potency and low affinity for cell-surface IFNAR and the poxvirus antagonist B18R

2018 ◽  
Vol 293 (41) ◽  
pp. 16057-16068 ◽  
Author(s):  
Bethany D. Harris ◽  
Jessica Schreiter ◽  
Marc Chevrier ◽  
Jarrat L. Jordan ◽  
Mark R. Walter
Keyword(s):  
Biological Activities ◽  
Human Interferon ◽  
Type I ◽  
Binding Affinities ◽  
Purification And Characterization ◽  
Cellular Assays ◽  
Mucosal Surfaces ◽  
Receptor Chain ◽  
The Common

IFNϵ and IFNκ are interferons that induce microbial immunity at mucosal surfaces and in the skin. They are members of the type-I interferon (IFN) family, which consists of 16 different IFNs, that all signal through the common IFNAR1/IFNAR2 receptor complex. Although IFNϵ and IFNκ have unique expression and functional properties, their biophysical properties have not been extensively studied. In this report, we describe the expression, purification, and characterization of recombinant human IFNϵ and IFNκ. In cellular assays, IFNϵ and IFNκ exhibit ∼1000-fold lower potency than IFNα2 and IFNω. The reduced potency of IFNϵ and IFNκ are consistent with their weak affinity for the IFNAR2 receptor chain. Despite reduced IFNAR2-binding affinities, IFNϵ and IFNκ exhibit affinities for the IFNAR1 chain that are similar to other IFN subtypes. As observed for cellular IFNAR2 receptor, the poxvirus antagonist, B18R, also exhibits reduced affinity for IFNϵ and IFNκ, relative to the other IFNs. Taken together, our data suggest IFNϵ and IFNκ are specialized IFNs that have evolved to weakly bind to the IFNAR2 chain, which allows innate protection of the mucosa and skin and limits neutralization of IFNϵ and IFNκ biological activities by viral IFN antagonists.

Isolation of FSH from bovine pituitary glands

1993 ◽  
Vol 137 (1) ◽  
pp. 59-68 ◽  
Author(s):  
J.-B. Wu ◽  
P. G. Stanton ◽  
D. M. Robertson ◽  
M. T. W. Hearn
Keyword(s):  
Amino Acid ◽  
Biological Activities ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
High Yield ◽  
Purification Procedure ◽  
In Vitro Bioassay ◽  
Α Subunit ◽  
Pituitary Glands

ABSTRACT An improved method is described for the isolation of FSH from bovine pituitary glands. The purification procedure consisted of an initial ammonium sulphate precipitation step followed by triazine-dye chromatography, immobilized metal affinity chromatography, high-performance anion-exchange chromatography and gel filtration. Three highly purified bovine FSH preparations (designated bFSH-A, -B and -C) were obtained, giving yields of approximately 5·7 mg FSH/kg bovine pituitary glands (wet weight), with specific radioreceptor activities for bFSH-A, -B and -C of 61, 25 and 29 units (NIH-FSH-S1)/mg protein respectively. The corresponding biological activities were 217 (bFSH-A), 62 (bFSH-B) and 86 (bFSH-C) units/mg, as measured by an FSH in-vitro bioassay. LH levels were found to be < 1% (w/w) as determined by an LH in-vitro bioassay. SDS-PAGE of these bFSH preparations under reducing conditions in 16% polyacrylamide gels showed two major silver-staining bands of apparent molecular masses 19·5 kDa and 15·8 kDa. Their amino acid compositions were in close agreement with the expected composition, based on the bFSH cDNA sequence and results reported by other investigators. N-terminal sequencing of the bFSH-A preparation yielded two major sequences consistent with α- and β-subunits, and a third minor (< 20%) sequence consistent with the α-subunit clipped at amino acid residue 6. It was concluded that the bFSH purification procedure reported here is a rapid method which produces bFSH in high yield and high purity, with radioreceptor and in-vitro specific activities comparable with those previously reported by other investigators. Journal of Endocrinology (1993) 137, 59–68

Immunochemical and biological characterization of calcitonin originating from transplanted medullary thyroid carcinoma in rats

1982 ◽  
Vol 99 (3) ◽  
pp. 397-403 ◽  
Author(s):  
Lisbeth Myhre ◽  
Arne Ekeland ◽  
Kaare M. Gautvik
Keyword(s):  
Biological Activities ◽  
Gel Filtration ◽  
Molecular Size ◽  
Serum Levels ◽  
Biologically Active ◽  
Subcutaneous Injections ◽  
Medullary Thyroid ◽  
Biological Potency ◽  
Medullary Thyroid Carcinomas

Abstract. The immunoreactive and biological activities of calcitonin (CT) produced by transplanted rat medullary thyroid carcinomas (MCT) have been studied. Immunoreactive CT (iCT) in serum and in MCT tissues of rats carrying tumours of generations 5–8 was characterized by gel filtration on Sephadex G-100 followed by radioimmunological measurements using region specific antiserum. The hypocalcaemic effect of sera and tumour extracts was tested in a rat bioassay. Rats with transplanted MCT of the 5th and 6th generations had mainly (70–84%) circulating iCT species of molecular size comparable to intact hormone. However, in rats with tumours of the 7th and 8th generations corresponding circulating iCT forms comprised less than 52% of total immunoreactivity while 32–38% eluted earlier. In conparison, iCT corresponding to intact hormone represented 30–50% of total immunoreactivity in the tumour extracts and no differences were observed between the generations. Subcutaneous injections of sera from MCT rats and of tumour extracts reduced the serum levels of ionized calcium in test rats. The sera containing mainly intact iCT showed the strongest biological potency. We conclude that rat MCT transplanted under the kidney capsule is able to secrete biologically active CT. However, the heterogeneity of circulating iCT increases in rats with transplanted tumours of older generations, approaching the heterogeneity of stored hormone in the gland.

scholarly journals Characterization of six recombinant human RNase H2 bearing Aicardi-Goutiéres syndrome causing mutations

2019 ◽  
Vol 166 (6) ◽  
pp. 537-545 ◽  
Author(s):  
Takuto Nishimura ◽  
Misato Baba ◽  
Saori Ogawa ◽  
Kenji Kojima ◽  
Teisuke Takita ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Enzyme Preparation ◽  
Gel Filtration ◽  
Wild Type ◽  
Melting Temperatures ◽  
Human Rnase ◽  
Accessory Subunits ◽  
A Subunit ◽  
Rnase H2

Abstract Mammalian RNase H2 is a heterotrimeric enzyme consisting of one catalytic subunit (A) and two accessory subunits (B and C). RNase H2 is involved in the removal of a single ribonucleotide embedded in genomic DNA and removal of RNA of RNA/DNA hybrids. In humans, mutation of the RNase H2 gene causes a severe neuroinflammatory disorder Aicardi-Goutières syndrome (AGS). Here, we examined the activity and stability of six recombinant human RNase H2 variants bearing one AGS-causing mutation, A-G37S (Gly37 in the A subunit is replaced with Ser), A-N212I, A-R291H, B-A177T, B-V185G, or C-R69W. The activity of A-G37S was 0.3–1% of that of the wild-type RNase H2 (WT), while those of other five variants were 51–120%. In circular dichroism measurement, the melting temperatures of variants were 50–53°C, lower than that of WT (56°C). These results suggested that A-G37S had decreased activity and stability than WT, while other five variants had decreased stability but retained activity. In gel filtration chromatography of the purified enzyme preparation, WT migrated as a heterotrimer, while A-R291H eluted in two separate peaks containing either the heterotrimer or only the A subunit, suggesting that some AGS-causing mutations affect the heterotrimer-forming stability of RNase H2.

Molecular characterization of four ADAMTS13 mutations responsible for congenital thrombotic thrombocytopenic purpura (Upshaw-Schulman syndrome)

2007 ◽  
Vol 98 (09) ◽  
pp. 593-599 ◽  
Author(s):  
Antoine Hommais ◽  
Julie Rayes ◽  
Anne Houllier ◽  
Bernadette Obert ◽  
Paulette Legendre ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Thrombotic Thrombocytopenic Purpura ◽  
Proteolytic Activity ◽  
Specific Activity ◽  
Electrophoretic Analysis ◽  
Full Length ◽  
Wild Type ◽  
Thrombocytopenic Purpura ◽  
Congenital Thrombotic Thrombocytopenic Purpura

SummaryADAMTS13 mutations S203P, R268P, R507Q and A596V were previously identified in French patients with hereditary thrombotic thrombocytopenic purpura (TTP) (Upshaw-Schulman syndrome). Mutated recombinant (r) ADAMTS13 were transiently expressed in COS-7 cells and characterized in comparison with wild-type (WT) rADAMTS13.ADAMTS13 antigen was qualitatively and quantitatively estimated by electrophoretic analysis and ELISA. Enzymatic activity was qualitatively and quantitatively estimated using GST-VWF73,FRETS-VWF73 fragments and full-length rVWF-WT as substrates. The four mutants and rADAMTS13-WT were present within the cells. Secretion level of rADAMTS13-WT reached 1,200 ng/ml. The four mutations strongly altered the secretion and biological activity of rADAMTS13. The percentage secretion was 21, 38 and 17% for rADAMTS13-S203P, -R268P and -A596V compared with rADAMTS13- WT. rADAMTS13-R507Q concentration was under the detection limit of the assay. In the four cases, no enzymatic activity was detected. After concentration, we confirmed that mutations S203P and R268P totally abolished the proteolytic activity of ADAMTS13. Due to the very low protease concentration, activity of rADAMTS13-R507Q was below the threshold of the assays. rADAMTS13-A596V had no proteolytic activity towards the full-length rVWF-WT whereas it exhibited a decreased specific activity of about 30% of that of rADAMTS13- WT towards FRETS-VWF73 fragment. Binding study of mutated rADAMTS13-S203P, -R268P and -A596V showed that the three mutations strongly decreased the interaction of ADAMTS13 with VWF. In conclusion, the four mutations, which led to a secretion defect, a loss of enzymatic activity and a decreased binding to the substrate, are responsible for the hereditary TTP in patients.

Construction and Characterization of Novel Staphylokinase Variants with Antiplatelet Aggregation Activity and Reduced Immunogenecity

2004 ◽  
Vol 36 (5) ◽  
pp. 336-342 ◽  
Author(s):  
Hua-Bo Su ◽  
Yu-Gao Zhang ◽  
Jin-Tian He ◽  
Wei Mo ◽  
Yan-Ling Zhang ◽  
...  
Keyword(s):  
Fibrinolytic Activity ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Final Concentration ◽  
Wild Type ◽  
E Coli ◽  
Antiplatelet Aggregation ◽  
Aggregation Activity

Abstract To develop target thrombolytic agents with fibrinolytic activity, antiplatelet aggregation activity and reduced immunogenicity, two staphylokinase variants containing Arg-Gly-Asp (RGD) motif were constructed. Gene expression was induced in E. coli JF1125 and the variants, designated DGR and RL1, were purified with gel filtration and ion-exchange chromatography and the purity was over 95%. The fibrinolytic activity and kinetic constants of the two variants were comparable to those of recombinant wild-type staphylokinase. Both the variants can inhibit the platelet aggregation at a final concentration of 2 μM. The titers of antibodies against variants were much lower than those against recombinant staphylokinase in guinea pigs, which indicated that the immunogenicity of the variants was greatly reduced. These results confirm that it is possible to design and produce a bifunctional protein that possesses fibrinolytic and antiplatelet aggregation activities.

scholarly journals Characterization of proinsulin- and proglucagon-converting activities in isolated islet secretory granules.

1981 ◽  
Vol 90 (2) ◽  
pp. 312-322 ◽  
Author(s):  
D J Fletcher ◽  
J P Quigley ◽  
G E Bauer ◽  
B D Noe
Keyword(s):  
Secretory Granules ◽  
Gel Filtration ◽  
Electrophoretic Analysis ◽  
Cleavage Sites ◽  
Ph Optimum ◽  
Diisopropyl Fluorophosphate ◽  
Granule Membrane ◽  
Soluble Components

The conversion of proglucagon and proinsulin by secretory granules isolated from both prelabeled and unlabeled anglerfish islets was investigated. Either granules isolated from tissue labeled with [3H]tryptophan and [14C]isoleucine or [35S]cysteine, or lysed granules from unlabeled tissue to which exogenously labeled prohormones had been added were incubated under various conditions. Acetic acid extracts of these granule preparations were analyzed for prohormone and hormone content by gel filtration. Both prelabeled and lysed, unlabeled secretory granules converted radiolabeled precursor peptides (Mr 8,000-15,000) to labeled insulin and glucagon. The accuracy of the cleavage process was established by demonstrating comigration of products obtained from in vitro cleavage with insulin and glucagon extracted from intact islets using electrophoresis and high-pressure liquid chromatography (HPLC). The pH optimum for granule-mediated conversion was found to be in the range of pH 4.5-5.5. Conversion of both proglucagon and proinsulin by secretory granules was significantly inhibited in the presence of antipain, leupeptin, p-chloromercuribenzoate (PCMB) or dithiodipyridine (DDP) but not chloroquine, diisopropyl fluorophosphate, EDTA, p-nitrophenyl guanidinobenzoate, soybean trypsin inhibitor, or N-p-tosyl-L-lysine chloromethyl ketone HCl. The inhibitory action of PCMB and DDP was reversed in the presence of dithiothreitol. Both membranous and soluble components of the secretory granules possessed significant converting activity. HPLC and electrophoretic analysis of cleavage products demonstrated that the converting activities of the membranous and soluble components were indistinguishable. The amount of inhibition of proinsulin and proglucagon conversion caused by 600 micrograms/ml porcine proinsulin was significantly lower than that caused by the same concentration of unlabeled anglerfish precursor peptides. These results indicate that the proinsulin and proglucagon converting enzyme(s) in the anglerfish pancreatic islet is a unique intracellular thiol proteinase(s) that may be granule membrane-associated and may require the presence of prohormone sequences in addition to the dibasic residues at cleavage sites for substrate recognition and/or binding.

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