A spectrin-like protein from mouse brain membranes: phosphorylation of the 235,000-dalton subunit

A mouse brain spectrin-like protein, which was an immunoreactive analogue of erythrocyte spectrin, has been isolated from demyelinated membranes. This spectrin analogue was a 10.5 S, 972,000 molecular weight (Mr) (alpha beta)2 tetramer containing subunits of 240,000 (alpha) and 235,000 (beta) Mr. We demonstrated that in vivo only the 235,000 Mr beta subunit of the mouse brain spectrin-like protein was phosphorylated, which was an analogous situation to mouse erythrocyte spectrin in which only the 220,000 Mr beta subunit was phosphorylated. Incubation of isolated membrane fractions with [gamma-32P]ATP +/- adenosine 3',5'-cyclic monophosphate (cAMP) indicated that mouse brain spectrin-like protein, mouse erythrocyte spectrin, and human erythrocyte spectrin's beta subunits were all phosphorylated in vitro by membrane-associated cAMP-independent protein kinases.

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Investigations into the post-translational modification and mechanism of isopenicillin N:acyl-CoA acyltransferase using electrospray mass spectrometry

Biochemical Journal ◽

10.1042/bj2940357 ◽

1993 ◽

Vol 294 (2) ◽

pp. 357-363 ◽

Cited By ~ 19

Author(s):

R T Aplin ◽

J E Baldwin ◽

P L Roach ◽

C V Robinson ◽

C J Schofield

Keyword(s):

Mass Spectrometry ◽

Electrospray Mass Spectrometry ◽

Beta Subunit ◽

Alpha Subunit ◽

British Library ◽

Beta Subunits ◽

Post Translational Modification ◽

Acetic Acids

Electrospray mass spectrometry (e.s.m.s.) was used to confirm the position of the post-translational cleavage of the isopenicillin N:acyl-CoA acyltransferase preprotein to give the alpha- and beta-subunits. The e.s.m.s. studies suggested partial modification of the alpha-subunit in vivo by exogenously added substituted acetic acids. E.s.m.s. has also allowed the observation in vitro of the transfer of the acyl group from several acyl-CoAs to the beta-subunit. N.m.r. data for the CoA species have been deposited as Supplementary Publication SUP 500173 (2 pages) at the British Library Document Supply Centre (DSC), Boston Spa, Wetherby, West Yorkshire LS23 7BQ, from whom copies can be obtained on the terms indicated in Biochem. J. (1993) 289, 9.

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The use of naturally occurring hybrid variants of chloramphenicol acetyltransferase to investigate subunit contacts

Biochemical Journal ◽

10.1042/bj1930541 ◽

1981 ◽

Vol 193 (2) ◽

pp. 541-552 ◽

Cited By ~ 7

Author(s):

L C Packman ◽

W V Shaw

Keyword(s):

Chloramphenicol Acetyltransferase ◽

Alpha Subunit ◽

Type I ◽

Chloramphenicol Resistance ◽

Type Iii ◽

Naturally Occurring ◽

Alpha Beta ◽

Beta 2

1. Hybrids of the tetrameric enzyme chloramphenicol acetyltransferase (EC 2.3.1.28) were formed in vivo in a strain of Escherichia coli which harbours two different plasmids, each of which normally confers chloramphenicol resistance and specifies an easily distinguished enzyme variant (type I or type III) which is composed of identical subunits. Cell-free extracts of the dual-plasmid strain were found to contain five species of active enzyme, two of which were the homomeric enzymes corresponding to the naturally occurring tetramers of the type-I (beta 4) and type-III (alpha 4) enzymes. The other three variants were judged to be the heteromeric hybrid variants (alpha 3 beta, alpha 2 beta 2, alpha beta 3). 2. The alpha 3 beta and alpha 2 beta 2 hybrids of chloramphenicol acetyltransferase were purified to homogeneity by combining the techniques of affinity and ion-exchange chromatography. The alpha beta 3 variant was not recovered and may be unstable in vitro. 3. The unique lysine residues that could not be modified with methyl acetimidate in each of the native homomeric enzymes were also investigated in the heteromeric tetramers. 4. Lysine-136 remains buried in each beta subunit of the parental (type I) enzyme and in each of the hybrid tetramers. Lysine-38 of each alpha subunit is similarly unreactive in the native type-III chloramphenicol acetyltransferase (alpha 4), but in the alpha 2 beta 2 hybird lysine-38 of each alpha subunit is fully exposed to solvent. Another lysine residue, fully reactive in the alpha 4 enzyme, was observed to be inaccessible to modification in the symmetrical hybrid. The results obtained for the alpha 3 beta enzyme suggest that lysine-38 in two subunits and a different lysine group (that identified in the alpha 2 beta 2 enzyme) in the third alpha subunit are buried. 5. A tentative model for the subunit interactions of chloramphenicol acetyltransferase is proposed on the basis of the results described.

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Expression of an in vitro biologically active equine LH/CG without C-terminal peptide (CTP) and/or beta26-110 disulphide bridge

Journal of Endocrinology ◽

10.1677/joe.0.1670117 ◽

2000 ◽

Vol 167 (1) ◽

pp. 117-124 ◽

Cited By ~ 9

Author(s):

C Galet ◽

M Chopineau ◽

N Martinat ◽

Y Combarnous ◽

F Guillou

Keyword(s):

Terminal Region ◽

Biologically Active ◽

Beta Subunit ◽

Disulphide Bond ◽

Beta Subunits ◽

Single Chain ◽

Disulphide Bridge ◽

Double Mutation

The C-terminal region of the beta subunit of the human chorionic gonadotrophin (hCG) is implied in heterodimer stability (beta26-110 disulphide bridge), in vitro LH bioactivity (region beta102-110) and in in vivo LH bioactivity (beta CTP). Like the hCG beta, the equine eLH and eCG beta subunits, also possess a C-terminal extension (CTP). But, in contrast to hCG, eLH and eCG bind to both LH and FSH receptors in species other than the horse. This allows investigation of the roles of the beta subunit C-terminal region of a eLH/CG recombinant molecule on both LH and FSH activities. To do so, the CTP was deleted and/or the beta26-110 disulphide bond was mutated and the resulting mutated beta subunits were transiently co-expressed with common alpha subunit in COS7 cells. These regions were also deleted in a betaalphaeLH/CG single chain also expressed in COS7 cells. The hormones produced were characterized by different ELISAs and in vitro LH and FSH bioassays. Mutation of the 26-110 disulphide bond and deletion of the betaCTP led to a decrease in eLH/CG heterodimer production. Double mutation promoted an additive effect on production of the heterodimer and of the corresponding tethered eLH/CG. The elimination of the beta26-110 disulphide bond in the betaalpha single chain had no effect on its production. However, neither the 26-110 disulphide bond nor the CTP mutations affected dimer stability and bioactivities of the secreted heterodimers and/or single chain molecules. Therefore, in contrast to hCG, the 26-110 S-S bond of the recombinant eLH/CG beta subunit does not seem to be essential for eLH/CG dimer stability upon secretion and expressing LH and FSH bioactivities.

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Alpha/Beta and Gamma Interferons Are Induced by Infection with Noncytopathic Bovine Viral Diarrhea Virus In Vivo

Journal of Virology ◽

10.1128/jvi.76.2.923-927.2002 ◽

2002 ◽

Vol 76 (2) ◽

pp. 923-927 ◽

Cited By ~ 43

Author(s):

B. Charleston ◽

L. S. Brackenbury ◽

B. V. Carr ◽

M. D. Fray ◽

J. C. Hope ◽

...

Keyword(s):

Bovine Viral Diarrhea Virus ◽

Transforming Growth Factor ◽

Bovine Viral Diarrhea ◽

Diarrhea Virus ◽

Viral Diarrhea ◽

Alpha Beta ◽

Interferon Responses ◽

Viral Diarrhea Virus

ABSTRACT In contrast to the results of previous in vitro studies, experimental infection of calves with noncytopathic bovine viral diarrhea virus (ncpBVDV) was found to induce strong alpha/beta and gamma interferon responses in gnotobiotic animals. These responses were associated with depressed levels of transforming growth factor β (TGF-β) in serum. The results of this study indicate that the immunosuppression caused by ncpBVDV is not associated with low interferon responses or elevated levels of TGF-β.

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The distribution of beta-1- and beta-2-adrenergic receptors of normal and reeler mouse brain: An in vitro autoradiographic study

Neuroscience ◽

10.1016/0306-4522(87)90283-1 ◽

1987 ◽

Vol 23 (1) ◽

pp. 199-210 ◽

Cited By ~ 16

Author(s):

D. Lorton ◽

J.N. Davis

Keyword(s):

Mouse Brain ◽

Adrenergic Receptors ◽

Autoradiographic Study ◽

Beta 2

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Platelet-mediated proteolytic down regulation of the anticoagulant activity of protein S in individuals with haematological malignancies

Thrombosis and Haemostasis ◽

10.1160/th11-07-0457 ◽

2012 ◽

Vol 107 (03) ◽

pp. 468-476 ◽

Cited By ~ 12

Author(s):

Ilze Dienava-Verdoold ◽

Marina R. Marchetti ◽

Liane C. J. te Boome ◽

Laura Russo ◽

Anna Falanga ◽

...

Keyword(s):

Protein C ◽

Normal Values ◽

Anticoagulant Activity ◽

Activated Protein C ◽

Protein S ◽

Intact Protein ◽

The Impact ◽

Independent Protein

SummaryThe natural anticoagulant protein S contains a so-called thrombin-sensitive region (TSR), which is susceptible to proteolytic cleavage. We have previously shown that a platelet-associated protease is able to cleave protein S under physiological plasma conditions in vitro. The aim of the present study was to investigate the relation between platelet-associated protein S cleaving activity and in vivo protein S cleavage, and to evaluate the impact of in vivo protein S cleavage on its anticoagulant activity. Protein S cleavage in healthy subjects and in thrombocytopenic and thrombocythaemic patients was evaluated by immunological techniques. Concentration of cleaved and intact protein S was correlated to levels of activated protein C (APC)-dependent and APC-independent protein S anticoagulant activity. In plasma from healthy volunteers 25% of protein S is cleaved in the TSR. While in plasma there was a clear positive correlation between levels of intact protein S and both APC-dependent and APC-independent protein S anticoagulant activities, these correlations were absent for cleaved protein S. Protein S cleavage was significantly increased in patients with essential thrombocythaemia (ET) and significantly reduced in patients with chemotherapy-induced thrombocytopenia. In ET patients on cytoreductive therapy, both platelet count and protein S cleavage returned to normal values. Accordingly, platelet transfusion restored cleavage of protein S to normal values in patients with chemotherapy-induced thrombocytopenia. In conclusion, proteases from platelets seem to contribute to the presence of cleaved protein S in the circulation and may enhance the coagulation response in vivo by down regulating the anticoagulant activity of protein S.

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Up-regulated expression of genes encoding Hrk and IL-3R beta subunit by TCDD in vivo and in vitro

Toxicology Letters ◽

10.1016/s0378-4274(01)00470-2 ◽

2002 ◽

Vol 129 (1-2) ◽

pp. 1-11 ◽

Cited By ~ 4

Author(s):

Joo-Hung Park ◽

Soo-Woong Lee

Keyword(s):

Beta Subunit ◽

Expression Of Genes ◽

Regulated Expression ◽

Genes Encoding

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Cannabinoid effects on adenylate cyclase and phosphodiesterase activities of mouse brain

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y77-125 ◽

1977 ◽

Vol 55 (4) ◽

pp. 934-942 ◽

Cited By ~ 10

Author(s):

Thomas W. Dolby ◽

Lewis J. Kleinsmith

Keyword(s):

Cyclic Amp ◽

Adenylate Cyclase ◽

Mouse Brain ◽

Biogenic Amine ◽

Specific Activity ◽

Intraperitoneal Administration ◽

The Brain

The experiments presented in this paper examine the mechanisms underlying the ability of cannabinoids to alter the in vivo levels of cyclic adenosine 3′,5′-monophosphate (cyclic AMP) in mouse brain. It was found that changes in cyclic AMP levels are a composite result of direct actions of cannabinoids on adenylate cyclase (EC 4.6.1.1) activity and indirect actions involving the potentiation or inhibition of biogenic amine induced activity of adenylate cyclase. Furthermore, the long-term intraperitoneal administration of 1-(−)-Δ-tetrahydrocannabinol to mice produced a form of phosphodiesterase (EC 3.1.4.17) in the brain whose activity is not stimulated by Ca2+, although its basal specific activity is similar to that of control animals. In vitro, the presence of the cannabinoids caused no significant changes in activity of brain PDE at the concentrations tested. Some correlations are presented which imply that many of the observed behavioral and physiological actions of the cannabinoids in mammalian organisms may be mediated via cyclic AMP mechanisms.

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In vitro and In vivo Assessment of Suitable Reference Region and Kinetic Modelling for the mGluR1 Radioligand [11C]ITDM in Mice

Molecular Imaging and Biology ◽

10.1007/s11307-019-01435-1 ◽

2019 ◽

Vol 22 (4) ◽

pp. 854-863 ◽

Cited By ~ 4

Author(s):

Daniele Bertoglio ◽

Jeroen Verhaeghe ◽

Špela Korat ◽

Alan Miranda ◽

Leonie wyffels ◽

...

Keyword(s):

Mouse Brain ◽

Pet Imaging ◽

Metabotropic Glutamate Receptor ◽

Kinetic Modelling ◽

Input Function ◽

Dependent Manner ◽

Reference Region ◽

Suitable Reference

Abstract Purpose This study aimed at investigating binding specificity, suitability of reference region-based kinetic modelling, and pharmacokinetics of the metabotropic glutamate receptor 1 (mGluR1) radioligand [11C]ITDM in mice. Procedures We performed in vivo blocking as well as displacement of [11C]ITDM during positron emission tomography (PET) imaging using the specific mGluR1 antagonist YM-202074. Additionally, we assessed in vitro blocking of [3H]ITDM at two different doses of YM-202074. As an alternative to reference region models, we validated the use of a noninvasive image-derived input function (IDIF) compared to an arterial input function measured with an invasive arteriovenous (AV) shunt using a population-based curve for radiometabolite correction and characterized the pharmacokinetic modelling of [11C]ITDM in the mouse brain. Finally, we also assessed semi-quantitative approaches. Results In vivo blocking with YM-202074 resulted in a decreased [11C]ITDM binding, ranging from − 35.8 ± 8.0 % in pons to − 65.8 ± 3.0 % in thalamus. Displacement was also markedly observed in all tested regions. In addition, in vitro [3H]ITDM binding could be blocked in a dose-dependent manner. The volume of distribution (VT) based on the noninvasive IDIF (VT (IDIF)) showed excellent agreement with the VT values based on the metabolite-corrected plasma input function regardless of the metabolite correction (r2 > 0.943, p < 0.0001). Two-tissue compartmental model (2TCM) was found to be the preferred model and showed optimal agreement with Logan plot (r2 > 0.960, p < 0.0001). A minimum scan duration of 80 min was required for proper parameter estimation. SUV was not reliable (r2 = 0.379, p = 0.0011), unlike the SUV ratio to the SUV of the input function, which showed to be a valid approach. Conclusions No suitable reference region could be identified for [11C]ITDM as strongly supported by in vivo and in vitro evidence of specific binding in all brain regions. However, by applying appropriate kinetic models, [11C]ITDM PET imaging represents a promising tool to visualize mGluR1 in the mouse brain.

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