A scaffold-associated DNA region is located downstream of the pea plastocyanin gene.

Chromosomal scaffold-associated DNA has been isolated from pea leaf nuclei treated with lithium diiodosalicylate to remove histones and then digested with restriction enzymes to remove the DNA in chromosomal loops. A scaffold-associated region (SAR) of DNA has been identified 8 to 9 kb downstream of the single-copy pea plastocyanin gene in proximity to a repetitive sequence present in 300 copies in the pea haploid genome. Isolated restriction fragments from within the SAR can bind to scaffold preparations in a binding assay in vitro. The nucleotide sequence of the SAR indicates a 540-bp 77% A+T-rich region containing many sequence elements in common with SARs from other organisms. Sequences with homology to topoisomerase II binding sites, A-box and T-box sequences, and replication origins are present within this AT-rich region.     

S-(N-methylcarbamoyl)glutathione: a reactive S-linked metabolite of methyl isocyanate

S-(N-methylcarbamoyl)glutathione, a chemically-reactive glutathione conjugate, has been isolated from the bile of rats administered methyl isocyanate and characterized, as its N-benzyloxycarbonyl dimethylester derivative, by tandem mass spectrometry. The ability of this glutathione adduct to donate an N-methylcarbamoyl moiety to the free -SH group of cysteine was evaluated in vitro with the aid of a highly specific thermospray LC/MS assay procedure. The glutathione adduct reacted readily with cysteine in buffered aqueous media (pH 7.4, 37 degrees C) and after 2 hr, 42.5% of the substrate existed in the form of S-(N-methylcarbamoyl)cysteine. The reverse reaction, i.e. between the cysteine adduct and free glutathione, also took place readily under these conditions. It is concluded that conjugation of methyl isocyanate with glutathione in vivo affords a reactive S-linked product which displays the potential to carbamoylate nucleophilic amino acids. The various systemic toxicities associated with exposure of animals or humans to methyl isocyanate could therefore be due to release of the isocyanate from its glutathione conjugate, which thus may serve as a vehicle for the transport of methyl isocyanate in vivo.     

Identification of a new cross-link and unique histidine adduct from bovine serum albumin incubated with malondialdehyde

Malondialdehyde, acetaldehyde, acrolein, and 4-hydroxynonenal are all products of fatty acid oxidation found in the fatty streaks of atherosclerotic arteries due to a lack of antioxidants and an increase in glycation products. Previously identified cross-links derived from these molecules have nearly always required more than one molecule of each type, although this is physiologically less likely than a reaction involving a single molecule. Here we provide indirect but strong evidence for a malondialdehyde-derived cross-link requiring just one malondialdehyde molecule to link arginine and lysine, giving 2-ornithinyl-4-methyl(1epsilon-lysyl)1,3-imidazole following a 4-day incubation of albumin with 8 mm malondialdehyde. This cross-link was identified as its partial degradation product Nepsilon-(2-carboxyl,2-aminoethane)-Nepsilon-methanoyl-lysine by NMR and mass spectrometry. Analysis of plasma from treated diabetic patients revealed that one patient levels had as high as 0.46%, 0.67% of their lysine/arginine residues modified by this cross-link, although others had lower levels. Alkaline hydrolysis of serum albumin also revealed two acid-labile malondialdehyde adducts of histidine in significant quantities, the isomers 4- and 2-ethylidene-histidine. These constituted up to 0.93% of the histidines in treated diabetic patients. Although collagen is readily cross-linked by malondialdehyde, none of these particular products could be found in incubations of collagen with malondialdehyde.     

Integrin activation state determines selectivity for novel recognition sites in fibrillar collagens

Only three recognition motifs, GFOGER, GLOGER, and GASGER, all present in type I collagen, have been identified to date for collagen-binding integrins, such as alpha(2)beta(1). Sequence alignment was used to investigate the occurrence of related motifs in other human fibrillar collagens, and located a conserved array of novel GER motifs within their triple helical domains. We compared the integrin binding properties of synthetic triple helical peptides containing examples of such sequences (GLSGER, GMOGER, GAOGER, and GQRGER) or the previously identified motifs. Recombinant inserted (I) domains of integrin subunits alpha(1), alpha(2) and alpha(11) all bound poorly to all motifs other than GFOGER and GLOGER. Similarly, alpha(2)beta(1) -containing resting platelets adhered well only to GFOGER and GLOGER, while ADP-activated platelets, HT1080 cells and two active alpha(2)I domain mutants (E318W, locked open) bound all motifs well, indicating that affinity modulation determines the sequence selectivity of integrins. GxO/SGER peptides inhibited platelet adhesion to collagen monomers with order of potency F >/= L >/= M > A. These results establish GFOGER as a high affinity sequence, which can interact with the alpha(2)I domain in the absence of activation and suggest that integrin reactivity of collagens may be predicted from their GER content.     

Multicenter phase 1/2 application of adenovirus-specific T cells in high-risk pediatric patients after allogeneic stem cell transplantation

Background

Adenovirus (ADV) reactivation can cause significant morbidity and mortality in children after allogeneic stem cell transplantation. Antiviral drugs can control viremia, but viral clearance requires recovery of cell-mediated immunity.

The ATPase Cycle of PcrA Helicase and Its Coupling to Translocation on DNA

The superfamily 1 bacterial helicase PcrA has a role in the replication of certain plasmids, acting with the initiator protein (RepD) that binds to and nicks the double-stranded origin of replication. PcrA also translocates single-stranded DNA with discrete steps of one base per ATP hydrolyzed. Individual rate constants have been determined for the DNA helicase PcrA ATPase cycle when bound to either single-stranded DNA or a double-stranded DNA junction that also has RepD bound. The fluorescent ATP analogue 2′(3′)-O-(N-methylanthraniloyl)ATP was used throughout all experiments to provide a complete ATPase cycle for a single nucleotide species. Fluorescence intensity and anisotropy stopped-flow measurements were used to determine rate constants for binding and release. Quenched-flow measurements provided the kinetics of the hydrolytic cleavage step. The fluorescent phosphate sensor MDCC-PBP was used to measure phosphate release kinetics. The chemical cleavage step is the rate-limiting step in the cycle and is essentially irreversible and would result in the bound ATP complex being a major component at steady state. This cleavage step is greatly accelerated by bound DNA, producing the high activation of this protein compared to the protein alone. The data suggest the possibility that ADP is released in two steps, which would result in bound ADP also being a major intermediate, with bound ADP·P_i being a very small component. It therefore seems likely that the major transition in structure occurs during the cleavage step, rather than P_i release. ATP rebinding could then cause reversal of this structural transition. The kinetic mechanism of the PcrA ATPase cycle is very little changed by potential binding to RepD, supporting the idea that RepD increases the processivity of PcrA by increasing affinity to DNA rather than affecting the enzymatic properties per se.     

Identification of MarvelD3 as a tight junction-associated transmembrane protein of the occludin family

Background

Protein translocation across the membrane of the Endoplasmic Reticulum (ER) is the first step in the biogenesis of secretory and membrane proteins. Proteins enter the ER by the Sec61 translocon, a proteinaceous channel composed of three subunits, α, β and γ. While it is known that Sec61α forms the actual channel, the function of the other two subunits remains to be characterized.

Results

In the present study we have investigated the function of Sec61β in Drosophila melanogaster. We describe its role in the plasma membrane traffic of Gurken, the ligand for the Epidermal Growth Factor (EGF) receptor in the oocyte. Germline clones of the mutant allele of Sec61β show normal translocation of Gurken into the ER and transport to the Golgi complex, but further traffic to the plasma membrane is impeded. The defect in plasma membrane traffic due to absence of Sec61β is specific for Gurken and is not due to a general trafficking defect.

Conclusion

Based on our study we conclude that Sec61β, which is part of the ER protein translocation channel affects a post-ER step during Gurken trafficking to the plasma membrane. We propose an additional role of Sec61β beyond protein translocation into the ER.     

Human Platelets Utilize Cycloxygenase-1 to Generate Dioxolane A3, a Neutrophil-activating Eicosanoid

Eicosanoids are important mediators of fever, pain, and inflammation that modulate cell signaling during acute and chronic disease. We show by using lipidomics that thrombin-activated human platelets generate a new type of eicosanoid that both stimulates and primes human neutrophil integrin (Mac-1) expression, in response to formylmethionylleucylphenylalanine. Detailed characterization proposes a dioxolane structure, 8-hydroxy-9,11-dioxolane eicosatetraenoic acid (dioxolane A₃, DXA₃). The lipid is generated in nanogram amounts by platelets from endogenous arachidonate during physiological activation, with inhibition by aspirin in vitro or in vivo, implicating cyclooxygenase-1 (COX). Pharmacological and genetic studies on human/murine platelets revealed that DXA₃ formation requires protease-activated receptors 1 and 4, cytosolic phospholipase A₂ (cPLA₂), Src tyrosine kinases, p38 MAPK, phospholipase C, and intracellular calcium. From data generated by purified COX isoforms and chemical oxidation, we propose that DXA₃ is generated by release of an intermediate from the active site followed by oxygenation at C8. In summary, a new neutrophil-activating platelet-derived lipid generated by COX-1 is presented that can activate or prime human neutrophils, suggesting a role in innate immunity and acute inflammation.     

Sequence of a cDNA for mouse thymidylate synthase reveals striking similarity with the prokaryotic enzyme

We report the nucleotide sequence of a cloned cDNA, pMTS-3, that contains a 1-kb insert corresponding to mouse thymidylate synthase (E.C. 2.1.1.45). The open reading frame of 921 nucleotides from the first AUG to the termination codon specifies a protein with a molecular mass of 34,962 daltons. The predicted amino acid sequence is 90% identical with that of the human enzyme. The mouse sequence also has an extremely high degree of similarity (as much as 55% identity) with prokaryotic thymidylate synthase sequences, indicating that thymidylate synthase is among the most highly conserved proteins studied to date. The similarity is especially pronounced (as much as 80% identity) in the 44-amino-acid region encompassing the binding site for deoxyuridylic acid. The cDNA sequence also suggests that mouse thymidylate synthase mRNA lacks a 3' untranslated region, since the termination codon, UAA, is followed immediately by a poly(A) segment.      

Interaction of calmodulin with phospholamban and caldesmon: comparative studies by 1H-NMR spectroscopy.

In order to identify comparative aspects of the interaction of calmodulin with its target proteins, proton magnetic-resonance studies of complex formation between calmodulin and defined segments of phospholamban and caldesmon have been undertaken. Residues 3-15 in the cytoplasmic region of phospholamban, an integral membrane protein of cardiac sarcoplasmic reticulum believed to regulate the calcium pumping ATPase, are shown to contribute to interaction with calmodulin. Using wheat germ calmodulin specifically modified with a spin-label to provide the spectral means for spatial localisation, these residues of phospholamban were correlated with binding in the vicinity of the probe attached to Cys-27 in the N-terminal domain of calmodulin. This interaction, relevant to the mechanism of calmodulin-dependent phosphorylation of phospholamban that relieves its inhibitory influence on the calcium pump, provides a useful model system for comparative study of the properties of calmodulin-binding domains. We contrast here a calmodulin-binding segment in the C-terminal region of caldesmon localised by 1H-NMR study of the interface(s) between the two proteins. These observations are discussed in the context of other calmodulin-binding sequences.