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Covalently closed circular DNA (cccDNA) forms a template for the replication of hepatitis B virus (HBV) and duck HBV (DHBV). Recent studies suggest that activation-induced cytidine deaminase (AID) functions in innate immunity, although its molecular mechanism of action remains unclear, particularly regarding HBV restriction. Here we demonstrated that overexpression of chicken AID caused hypermutation and reduction of DHBV cccDNA levels. Inhibition of uracil-DNA glycosylase (UNG) by UNG inhibitor protein (UGI) abolished AID-induced cccDNA reduction, suggesting that the AID/UNG pathway triggers the degradation of cccDNA via cytosine deamination and uracil excision., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

The Ubr1-like canonical N-recognins, widely conserved ubiquitin ligases in eukaryotes, play a role in the N-end rule pathway-mediated degradation of substrates harboring basic (type-1) or bulky hydrophobic (type-2) amino acids at the N-terminus. In this study, the roles of conserved domains were studied in the Schizosaccharomyces pombe Ubr11 protein. Mutations in the UBR box and the autoinhibitory domain blocked degradation of both type-1 and type-2 substrates, expression of peptide transporter genes, and the uptake of oligopeptides. An N-domain mutant was normal for the type-1-related function, but nevertheless failed to express peptide transporters. These data suggest the importance of the type-2-related activity of Ubr11 for its in vivo function., (Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Although the alpha-helical Y(X)4Lvarphi containing region of eIF4E-binding protein (4EBP) is the major binding region with eukaryotic initiation factor 4E (eIF4E), the roles of its N- and C-terminal regions in the binding are hardly known. To clarify the roles of these flexible regions in the interaction, the binding features of the sequentially N-, C-, or both-terminal-residue-deleted 4EBP2 mutants were investigated by surface plasmon resonance (SPR) analysis. It was shown that the C-terminal His74-Glu89 sequence has an auxiliary, but indispensable, function in stabilizing the binding to eIF4E. The possible interaction with eIF4E was estimated by molecular dynamics simulation. This is the first report on the importance of the C-terminal flexible region in the eIF4E-binding regulation of 4EBP.

We developed a transgenic (Tg) rat model that overexpresses human proadrenomedullin N-terminal 20 peptide (PAMP) only and then compared the effects of unilateral nephrectomy followed by a high salt diet for five weeks in Tg and wild-type rats. We found that systolic blood pressure was significantly lower in Tg UNX rats and cardiac hypertrophy and myocardial fibrosis was also attenuated in Tg rats. Evaluation of gene expression showed suppression of cardiac local renin-angiotensin system (RAS) in Tg rat. These results suggest that in addition to reducing blood pressure, PAMP suppresses cardiac hypertrophy through negative regulation of the local cardiac RAS.

Insertion and deletion analyses of a protein have been less common than point mutation analyses, partly due to the lack in effective methods. This is the case with the green fluorescent protein (GFP), which is so widely applied in molecular biology and other fields. In this paper we first introduce a systematic approach for generating insertion/deletion mutants of GFP. A new technology of Y-ligation-based block shuffling (YLBS) was successfully applied to produce size-altered GFPs, providing insertion-containing GFPs of fluorescence, though no deletion type of fluorescence was obtained so far as examined. The analysis of these proteins suggested that size alteration (deletion/insertion) is acceptable so far as some type of rearrangement in a local structure can accommodate it. This paper demonstrates that YLBS can generate insertion and deletion mutant libraries systematically, which are beneficial in the study of structure-function relationship.

MIST (mast cell immunoreceptor signal transducer; also termed Clnk) is an adaptor protein structurally related to SLP-76-family hematopoietic cell-specific adaptor proteins. We demonstrate here that two major MIST-associated phosphoproteins expressed in mast cell lines are SLAP-130 and SKAP55, adaptors known to interact with the Src-homology (SH) 2 domain of Src-family protein tyrosine kinases (PTKs). MIST directly associated with SLAP-130 via its SH2 domain, and collaboration of SLAP-130 with SKAP55 was required for the recruitment of MIST to Lyn. Furthermore, MIST was preferentially recruited to Fyn rather than Lyn, which is regulated by higher affinity binding of SLAP-130 and SKAP55 with the Fyn-SH2 domain than the Lyn-SH2 domain. Our results suggest that the MIST-SLAP-130-SKAP55 adaptor complex functions downstream of high-affinity IgE receptor-associated Src-PTKs in mast cells., (Copyright 2003 Federation of European Biochemical Societies)

When coexpressed with receptor activity-modifying protein (RAMP)2 or -3, calcitonin receptor-like receptor (CRLR) functions as an adrenomedullin (AM) receptor (CRLR/RAMP2 or -3). Coexpression of rat (r)CRLR with rRAMP deletion mutants in HEK293T cells revealed that deletion of residues 93-99 from rRAMP2 or residues 58-64 from rRAMP3 significantly inhibits high-affinity [125I]AM binding and AM-evoked cAMP production, despite full cell surface expression of the receptor heterodimer. Apparently, these two seven-residue segments are key determinants of high-affinity agonist binding to rAM receptors and of receptor functionality. Consequently, their deletion yields peptides that are able to serve as negative regulators of AM receptor function.

Proadrenomedullin N-terminal 20 peptide (PAMP-20) is a potent hypotensive peptide processed from the adrenomedullin (AM) precursor. We developed a specific radioimmunoassay which recognizes the C-terminal region of PAMP-20. Using this radioimmunoassay, the distribution of immunoreactive (ir-) PAMP was determined in porcine tissues. High concentrations of ir-PAMP were observed in the adrenal medulla and in the atrium, and these values were comparable to the corresponding concentrations of ir-AM. The concentration of ir-PAMP was almost the same as that of ir-AM in the kidney, while ir-PAMP was significantly lower than ir-AM in the ventricle, lung, and aorta. Reversed-phase high performance liquid chromatography in each porcine tissue sample revealed that two major peaks of ir-PAMP existed: one emerged at a position identical to that of authentic porcine PAMP-20; the other unknown peak was eluted earlier. The unknown peptide was purified to homogeneity from porcine adrenal medulla, and its complete amino acid sequence was determined. This peptide was found to be PAMP[9-20] with a C-terminal amide structure, and was named PAMP-12. Intravenous injections of PAMP-12 in anesthetized rats showed a significant hypotensive effect in a dose-dependent fashion, and the effect was comparable to that of PAMP-20. These data indicate that PAMP-12, a major component of ir-PAMP, is processed from the AM precursor, as is PAMP-20, and may participate in cardiovascular control.

Plastoquinone-9 (PQ-9)-depleted PSII reaction center core complex, consisting of CP47/D1/D2/Cytb-559/I, was isolated from spinach PSII particles. PQ-9, lipids and several proteins were extracted from the original PSII particles and separated by several steps of chromatography to be reconstituted into the isolated complex. PQ-9 reconstituted in the complex with the help of thylakoid lipids (digalactosyldiglyceride) did not function as QA by itself. However, PQ-9 simultaneously reconstituted with L protein and the thylakoid lipids successfully functioned as QA in the complex. Other proteins of PSII origin, such as CP43, H, K, nuclear encoded 4.1 and 5.0 kDa proteins, are unable to restore the QA activity in the complex.

Adrenomedullin is a new bioactive peptide recently isolated from pheochromocytoma. We report on the rat adrenomedullin distribution and molecular forms in various tissues and plasma. Using a sensitive radioimmunoassay system for rat adrenomedullin, high concentrations of immunoreactive rat adrenomedullin were detected in adrenal gland, lung and cardiac atrium. In lung and atrium, the immunoreactivity concentration in rat was about 6-10 times higher than that in human. The mean plasma concentration of immunoreactive rat adrenomedullin was 3.60 +/- 0.34 fmol/ml (mean +/- S.D.). Analysis in adrenal gland, lung and atrium with reverse-phase and gel-filtration high-performance liquid chromatography showed that most immunoreactive rat adrenomedullin emerged as a single peak at a position exactly identical to that of the authentic rat adrenomedullin peptide, synthesized according to the sequence predicted from the cDNA.

Proadrenomedullin N-terminal 20 peptide (PAMP) is a candidate for a novel biologically active peptide processed from an adrenomedullin precursor. Using a radioimmunoassay for human PAMP, major and minor immunoreactive PAMPs were purified from porcine adrenal medulla and complete amino acid sequences were determined. The major immunoreactive peptide was PAMP itself with an amidated carboxy terminus. The minor one was determined to be PAMP[5-20]. An intravenous bolus injection of human PAMP in anesthetized rats caused a rapid and strong hypotensive effect in a dose dependent manner. The present data indicate that PAMP is an endogenous biologically active peptide which is processed from adrenomedullin precursor.

Bovine cultured adrenal medullary cells (4 x 10(6)) contained 4266.5 +/- 370.0 fmol of immunoreactive adrenomedullin and 373.4 +/- 32.6 nmol of catecholamines. Nicotinic (but not muscarinic) receptors mediated the Ca(2+)-dependent co-secretion of adrenomedullin and catecholamines, with the molar ratio of adrenomedullin/catecholamines secreted into the medium being equal to the ratio stored in the cells. The concentration-response curve of carbachol for adrenomedullin secretion (EC50 42 microM) was similar to that for catecholamine secretion (EC50 63 microM). Reverse phase HPLC analysis showed that immunoreactive adrenomedullins in the cells and secreted into the medium were both eluted exclusively at the position almost identical to synthetic human adrenomedullin[1-52]NH2.

A specific and sensitive radioimmunoassay for adrenomedullin has been developed. Half-maximal inhibition of binding of radioiodinated adrenomedullin was observed at 4 fmol/tube. The radioimmunoassay recognized the entire adrenomedullin molecule and has little crossreactivity with adrenomedullin fragment peptides. Adrenomedullin-like immunoreactivity was found to circulate in human plasma at considerable concentration (3.3 +/- 0.39 fmol/ml). The immunoreactivity of adrenomedullin was eluted at almost the same position as synthetic adrenomedullin on gel-filtration chromatography and reverse-phase high-performance liquid chromatography, suggesting that circulating adrenomedullin recognized by the present radioimmunoassay is identical or very similar to authentic adrenomedullin. Plasma immunoreactive adrenomedullin significantly increased in patients with hypertension, with a progressive rise proportionate to disease severity.

Porcine adrenomedullin was isolated from adrenal medulla extract and its amino acid sequence was determined. The peptide is identical to human adrenomedullin with a single replacement of Gly for Asn at position 40. The cDNA clone encoding the porcine adrenomedullin precursor was isolated and sequenced. The precursor for adrenomedullin (preproadrenomedullin) is 188 amino acids in length, including the adrenomedullin sequence, followed by a glycine (the amide donor). In addition to adrenomedullin, proadrenomedullin (proAM) contains a candidate for a unique 20-residue peptide, proAM-N20, whose carboxy-terminus may be amidated. By RNA blot analysis, porcine adrenomedullin mRNA was found to be highly expressed in several porcine tissues including lung and kidney as well as adrenal medulla.

A specific and sensitive radioimmunoassay for human adrenomedullin has been developed and distribution and characterization of immunoreactive adrenomedullin in human tissue were investigated. The radioimmunoassay specifically recognizes its carboxyterminal region and half maximal inhibition of binding of radioiodinated adrenomedullin(40-52)NH2 was observed at 11 fmol/tube. Immunoreactive adrenomedullin was abundant in adrenal medulla (47.7 +/- 26.1 fmol/mg, mean +/- S.D.) and was ubiquitously found in all tissue examined. The mean plasma concentration of adrenomedullin in three normal individuals was 17.2 +/- 6.4 pg/ml (mean +/- S.D.). By analysis with reverse-phase high-performance liquid chromatography coupled with the radioimmunoassay, most immunoreactive adrenomedullin in the adrenal medulla, atrium and lung was found to be adrenomedullin(1-52)NH2.

Soybean seeds contain three lipoxygenase isozymes. The functions of these lipoxygenase isozymes in n-hexanal generation were investigated by using mutant lines which lack two or three isozymes. In the presence of linoleic acid, the level of n-hexanal produced was highest in the lipoxygenase-1, -3 double deficient line, followed by the lipoxygenase-2, -3 double deficient, wild type, and lipoxygenase-1, -2, -3 triple deficient lines in that order, and lowest in the lipoxygenase-1, -2 double deficient line. This suggests that lipoxygenase-3 itself cannot produce the n-hexanal precursor and inhibits the n-hexanal generation through other pathways.

The gene coding for oligo-1,6-glucosidase from Bacillus cereus ATCC7064 has been overexpressed in Escherichia coli MV1184 cells under the control of the lac promoter in the genetically engineered plasmid pBCE4-2. Oligo-1,6-glucosidase was purified in large quantities and was crystallized at 25 degrees C by using a hanging drop vapor diffusion method with 53% saturated ammonium sulfate. The crystals have the shape of hexagonal bipyramids and belong to the space group P6(2) or P6(4) with lattice constants of a = b = 106.1 A, c = 120.0 A and gamma = 120 degrees.

Based on the crystal structure of the papain-E-64-c complex, 3-dimensional binding modes of a series of epoxysuccinyl amino acid derivatives to the papain active site have been constructed and the structure-inhibitory activity relationship has been analyzed using the accessible surface area and nonbonded energy parameters. The result indicates the importance of the hydrophobic interaction between the amino acid side chain of the inhibitor and the papain Val-157 residue for revealing the potent inhibitory activity.

Simulations of the molecular dynamics of the [Met5]enkephalin monomer and dimer structures in water have been carried out. The dynamic trajectories have been analyzed in terms of the distances between intra- or intermolecular polar atoms. The time-correlated conformational transitions of an extended monomer structure have been converged into a stationary state among the beta-bend folded forms. However, the dynamics simulation of an extended antiparallel dimer structure has shown no noticeable conformation change. These results imply that both the beta-bend monomer and the extended dimer structures exist together as the fundamental conformation of enkephalins.

The three-dimensional structure of the E-64-c-papain complex has been determined by X-ray crystal analysis at 2.5 A resolution (conventional R = 26.9%). The structure determined indicates that: (i) the C2 atom of the oxirane ring of E-64-c is covalently bound by the S gamma atom of Cys-25 of papain; (ii) this covalent bond formation results in a configurational conversion of the oxirane C2 atom from the S- to the R-form; and (iii) extensive hydrogen bonding and hydrophobic interactions are responsible for the specific interaction of the E-64-c molecule with papain.