Through-bond correlation of adenine H2 and H8 protons in unlabeled DNA fragments by HMBC spectroscopy

Summary A new application of the HMBC experiment is presented that provides a useful means to discriminate between H2 and H8 proton resonances, to assign the base proton resonances to the various residue types and, most importantly, to correlate the H2 and H8 protons for adenine or inosine residues in natural abundance ¹³C fragments. The utility of this experiment is demonstrated for an unlabeled DNA 20-mer. Thanks to the obtained results, preliminary conclusions could be drawn regarding the molecular conformations of the non-canonical G/I-A base pairs in the hairpin formed by this fragment.     

In vitro anti- and pro-oxidative effects of natural polyphenols

The anti- and pro-oxidative effects of phenolic compounds and antioxidants were studied in two different in vitro model systems utilizing ethyl linoleate and 2′-deoxyguanosine (2′-dG) as oxidative substrates, and a Fenton reaction (H₂O₂, Fe²⁺) to initiate oxidation. Oxidation of the biomolecules in both model systems exhibited dose dependency. In the 2′-dG assay, oxidation was closely related to H₂O₂ generation, which occurred during autoxidation of the phenolics. Hydroxylating activity was greatly enhanced by Mn²⁺ and Cu²⁺, but not by Zn²⁺ or Co²⁺. Ethyl linoleate peroxidation was inhibited by low concentrations of catechol, quercitin, and instant coffee. However, peroxidation was promoted by high concentrations of the same compounds, probably by recycling of chelated inactive Fe³⁺ to the active Fe²⁺ state.     

Oral administration of antigen does not influence the proliferation and IFN-γ production of responsive CD8+ T cells but enables to establish T cell clones with different lymphokine production profile.

Feeding of a whole casein diet, which abolished the α_s1-casein-specific proliferation and IFN-γ productivity of CD⁴⁺ T cells, did not affect the proliferative response of CD8⁺ T cells with regard to the antigen dose response, cell dose response, kinetics of the proliferation and epitope specificity, as well as IFN-γ production. To assess the characteristics of the CD8⁺ T cells, we established α_s1-casein-specific CD8⁺ T cell clones from both casein-fed and control mice. The established clones produced different amount of IFN-γ and IL-10, and one clone derived from the casein-fed mice produced a remarkable amount of IL-10. The clones from casein-fed mice produced considerable amounts of TGF-β, while those from control mice produced only small amounts. The possible role of CD8⁺ T cells in oral tolerance is discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Analysis of clonality in cytologic material using the polymerase chain reaction (PCR)

Analysis of clonality in cytologic material using the polymerase chain reaction (PCR) Immunoglobulin heavy chain (IgH) gene rearrangement analysis was performed on 27 fine needle aspiration (FNA) specimens (13 reactive hyperplasia, 11 B cell non-Hodgkin's lymphoma (B-NHL), one Hodgkin's disease and two suspicious of non-Hodgkin's lymphoma). Satisfactory amplification was achieved in 23/27 cases. A polyclonal pattern was seen in 14 cases (11 reactive hyperplasia, one B-NHL, one suspicious of lymphoma, one Hodgkin's disease). A monoclonal band was seen in nine cases (eight B-NHL, one reactive hyperplasia). Amplification was unsuccessful in four cases. Clonal analysis by PCR-based IgH gene rearrangement analysis can be successfully applied to FNA material and can be useful in diagnosis, but the results must be interpreted in conjunction with morphology and other ancillary information. Analyse de la clonalité en utilisant la PCR sur matériel cytologique L'analyse des réarrangements géniques des chaînes lourdes des immunoglobulines (IgH) a été réalisée sur 27 produits de ponction à l'aiguille fine (13 cas d'hyperplasie réactionnelle, 11 cas de lymphome non hodgkinien de type B (LNH-B), un cas de maladie de Hodgkin et deux cas suspects de lymphome malin non hodgkinien. Une amplification satisfaisante a été obtenue dans 23 cas sur 27. Un aspect polychonal a été observé dans 14 cas (11 hyperplasies réactionnelles, 1 lymphome malin non hodgkinien de type B, 1 cas suspect de lymphome, 1 cas de maladie de Hodgkin). Une bande monoclonale a été observée dans 9 cas (8 LNH-B, 1 hyperplasie réactionnelle). L'amplification n'a pas réussi dans quatre cas. L'analyse de la clonalité par analyse des réarrangements géniques par PCR peut être appliquée avec succès au matériel cytologique qui est obtenu par ponction à l'aiguille fine et elle peut être utile au plan diagnostique, mais les résultats doivent étre interprétés en intégrant les informations morphologiques et les autres données complémentaires. Polymerase Chain Reaction an zytologischem Material Die Analyse schwerer Immunglobulinketten (IgH) wurde an 27 FNP (13 reaktive Hyperplasien, 11 B-NHL, 1 Hodgkin und und zwie verdächtige Fälle) analysiert. Eine ausreichende Amplifikation wurde in 23/27 Fällen erzielt. Ein polyklonales Muster wurde in 14 Fällen beobachtet (11 reaktive Hyperplasien, 1 B-NHL, 1 Hodgkin sowie 1 verdächtiger Fall). Ein monoklonales Band wurde 9 mal gefunden (8 B-NHL, 1 reaktive Hyperplasie). Die Amplifikation war ungenügend in 4 Fällen. Die PCR-Analyse kann für die Diagnostik von Nutzen sein muss jedoch zusammen mit der Morphologie und anderen Informationen interpretiert werden.     

Crystallization of the first three domains of the human insulin-like growth factor-1 receptor.

The insulin-like growth factor-1 receptor (IGF-1R) is a tyrosine kinase receptor of central importance in cell proliferation. A fragment (residues 1-462) comprising the L1-cysteine rich-L2 domains of the human IGF-1R ectodomain has been overexpressed in glycosylation-deficient Lec8 cells and has been affinity-purified via a c-myc tag followed by gel filtration. The fragment was recognized by two anti-IGF-1R monoclonal antibodies, 24-31 and 24-60, but showed no detectable binding of IGF-1 or IGF-2. Isocratic elution of IGF-1R/462 on anion-exchange chromatography reduced sample heterogeneity, permitting the production of crystals that diffracted to 2.6 A resolution with cell dimensions a = 77.0 A, b = 99.5 A, c = 120.1 A, and space group P2(1)2(1)2(1).     

Hydrogen exchange: the modern legacy of Linderstrøm-Lang.

This discussion, prepared for the Protein Society's symposium honoring the 100th anniversary of Kaj Linderstrøm-Lang, shows how hydrogen exchange approaches initially conceived and implemented by Lang and his colleagues some 50 years ago are contributing to current progress in structural biology. Examples are chosen from the active protein folding field. Hydrogen exchange methods now make it possible to define the structure of protein folding intermediates in various contexts: as tenuous molten globule forms at equilibrium under destabilizing conditions, in kinetic intermediates that exist for less than one second, and as infinitesimally populated excited state forms under native conditions. More generally, similar methods now find broad application in studies of protein structure, energetics, and interactions. This article considers the rise of these capabilities from their inception at the Carlsberg Labs to their contemporary role as a significant tool of modern structural biology.     

Domain Evolution in the α-Amylase Family

The available amino acid sequences of the α-amylase family (glycosyl hydrolase family 13) were searched to identify their domain B, a distinct domain that protrudes from the regular catalytic (β/α)₈-barrel between the strand β3 and the helix α3. The isolated domain B sequences were inspected visually and also analyzed by Hydrophobic Cluster Analysis (HCA) to find common features. Sequence analyses and inspection of the few available three-dimensional structures suggest that the secondary structure of domain B varies with the enzyme specificity. Domain B in these different forms, however, may still have evolved from a common ancestor. The largest number of different specificities was found in the group with structural similarity to domain B from Bacillus cereus oligo-1,6-glucosidase that contains an α-helix succeeded by a three-stranded antiparallel β-sheet. These enzymes are α-glucosidase, cyclomaltodextrinase, dextran glucosidase, trehalose-6-phosphate hydrolase, neopullulanase, and a few α-amylases. Domain B of this type was observed also in some mammalian proteins involved in the transport of amino acids. These proteins show remarkable similarity with (β/α)₈-barrel elements throughout the entire sequence of enzymes from the oligo-1,6-glucosidase group. The transport proteins, in turn, resemble the animal 4F2 heavy-chain cell surface antigens, for which the sequences either lack domain B or contain only parts thereof. The similarities are compiled to indicate a possible route of domain evolution in the α-amylase family. Received: 4 December 1996 / Accepted: 13 March 1997     

Distribution and Subcellular Localization of High-Molecular-Weight Microtubule-Associated Protein-2 Expressing Exon 8 in Brain and Spinal Cord

Abstract: The expression of high-molecular-weight (HMW) microtubule-associated protein-2 (MAP-2) expressing exon 8 (MAP-2+8) was examined by immunoblotting during rat brain development and in sections of human CNS. In rat brain, HMW MAP-2+8 expression was detected at embryonic day 21 and increased during postnatal development. In adult rats, HMW MAP-2+8 comigrated with MAP-2a. In human adult brain, HMW MAP-2+8 was expressed in select neuronal populations, including pyramidal neurons of layers III and V of the neocortex and parahippocampal cortex, pyramidal neurons in the endplate, CA2 and subiculum of the hippocampus, and the medium-sized neurons of the basal ganglia. In the cerebellum, a subpopulation of Golgi neurons in the internal granular cell layer and most Purkinje cells were also stained. In the spinal cord staining was observed in large neurons of the anterior horn. Staining was present in cell bodies and dendrites but not in axons. At the ultra-structural level, HMW MAP-2+8 immunoreactivity was observed on mitochondrial membranes and in postsynaptic densities (PSDs) of some asymmetric synapses in the midfrontal cortex and spinal cord. Immunoblots of proteins isolated from enriched mitochondrial and PSD fractions from adult human frontal lobe and rat brains confirmed the presence of HMW MAP-2+8. The presence of HMW MAP-2+8 in dendrites and in close proximity to PSDs supports a role in structural and functional attributes of select excitatory CNS synapses.     

Down-Regulation of T-Cell Proliferation in Response to Soluble Anti-CD3 Antibodies through Development of Redirected Cytolytic Activity Eliminating Costimulatory Cells

CD4⁺ T-depleted spleen cells (CD8⁺ T cells) activated by anti-CD3 antibodies (aCD3) suppressed proliferation of CD8⁺ T-depleted spleen cells (CD4⁺ T cells) and fresh normal T cells in response to aCD3. Antigen-nonspecific cytolytic activity was induced in splenic CD8⁺ T cells by stimulation with aCD3 and showed the peak level on day 3, whereas cytolytic activity induced in CD4⁺ T cells was weak. Intact Ig but not F(ab')₂ of aCD3 induced and mediated cytolytic activity. Correspondingly, the cytolytic activity induced by aCD3 was directed against target cells bearing Ig-binding Fc-receptor activity and cytolysis was inhibited by the addition of free Ig into the assay system. We showed that aCD3-activated T cells carried a high level of aCD3 on their surface at the time after the peak proliferation when they attained high cytolytic activity. This raised the possibility that the anti-CD3-induced aCD3-redirected cytolytic activity eliminated Fc-receptor-bearing costimulatory cells in the culture for down-regulation of the T-cell proliferation. This view was supported by partial restoration of anti-CD3-induced low responsiveness of CD8⁺ T cells by the addition of fresh costimulatory cells. These results suggested a new pathway of down-regulation of T-cell proliferation by aCD3-activated cytolytic CD8⁺ T cells.     

缩胆囊素和促胰液素对豚鼠离体胃平滑肌运动的影响

用８个肌槽同时记录豚鼠胃不同部位肌条的收缩活动,以观察八肽缩胆囊素（ＣＣＫ－８）和促胰液素的影响。结果表明：ＣＣＫ－８能（１）增高各部位纵行肌和环行肌的张力;（２）加快胃体纵行肌,胃窦纵行肌、环行肌和幽门环行肌的收缩频率;（３）增大胃窦环行肌收缩波平均振幅和（４）增加幽门环行肌收缩波运动指数,但减少胃体和胃窦纵行肌收缩波平均振幅。上述作用均不能被阿托品和消炎病所阻断。而促胰液素对各部位肌条的收缩活动没有明显的影响。