Thiol Reduction of 3′-Azidothymidine to 3′-Aminothymidine: Kinetics and Biomedical Implications

The ability of thiols to reduce 3-azidothymidine (AZT) to 3-aminothymidine has been investigated. Incubation with glutathione, dithiothreitol (DTT), or mercaptoethanol at pH 7.2 and 37°C leads to quantitative reduction of the azido moiety to an amine. The reaction is first order in AZT and first order in reducing agent (mono- or dithiol). The second-order rate constants are 2.77 × 10^–3, 6.55 × 10^–5, and 6.35 × 10^–6 M ^–l sec^–1 for the dithiothreitol, glutathione, and mercaptoethanol reductions, respectively. The thiol reduction of alkyl azide to amine under mild conditions is a synthetic method particularly suitable for water-soluble azido compounds that are sensitive to catalytic hydrogenation. The potential for the mono- or dithiol-mediated reduction of alkyl azides under biological conditions must be considered when conducting studies of azido drugs.     

The RNA helicase, nucleotide 5'-triphosphatase, and RNA 5'-triphosphatase activities of Dengue virus protein NS3 are Mg2+-dependent and require a functional Walker B motif in the helicase catalytic core

The nonstructural protein 3 (NS3) of Dengue virus (DV) is a multifunctional enzyme carrying activities involved in viral RNA replication and capping: helicase, nucleoside 5'-triphosphatase (NTPase), and RNA 5'-triphosphatase (RTPase). Here, a 54-kDa C-terminal domain of NS3 (DeltaNS3) bearing all three activities was expressed as a recombinant protein. Structure-based sequence analysis in comparison with Hepatitis C virus (HCV) helicase indicates the presence of a HCV-helicase-like catalytic core domain in the N-terminal part of DeltaNS3, whereas the C-terminal part seems to be different. In this report, we show that the RTPase activity of DeltaNS3 is Mg2+-dependent as are both helicase and NTPase activities. Mutational analysis shows that the RTPase activity requires an intact NTPase/helicase Walker B motif in the helicase core, consistent with the fact that such motifs are involved in the coordination of Mg2+. The R513A substitution in the C-terminal domain of DeltaNS3 abrogates helicase activity and strongly diminishes RTPase activity, indicating that both activities are functionally coupled. DV RTPase seems to belong to a new class of Mg2+-dependent RTPases, which use the active center of the helicase/NTPase catalytic core in conjunction with elements in the C-terminal domain.     

A comparative study of the proteolytic enzymes of Trypanosoma brucei, T. equiperdum, T. evansi, T. vivax, Leishmania tarentolae and Crithidia fasciculata

Four types of proteolytic activity were detected in the bloodstream form of each of the four Trypanosoma species: (i) HPAase, active on hide powder azure and detected on polyacrylamide gels containing denatured haemoglobin; (ii) AZCase, active on azocasein; (iii) type 1, active on the chromogenic peptide N-benzoyl-L-prolyl-L-phenylalanyl-L-arginine p-nitroanilide in the presence of dithiothreitol, and (iv) type 2, active against several nitroanilide derivatives in the absence of dithiothreitol. Studies of the pH optimum, dithiothreitol requirement and inhibitor sensitivities of the proteolytic activities suggested that: (a) HPAase and type 1 activities could be due to the same enzymes, probably a family of cysteine proteinases; (b) AZCase had some characteristics of a cysteine proteinase, but was not identical to HPAase, and (c) type 2 activity could be due to a serine proteinase. Procyclic T. brucei contained relatively low cysteine proteinase activities (HPAase, AZCase and type 1) but high type 2 activity. Their proteolytic enzymes thus were apparently more similar to those in Crithidia fasciculata and Leishmania tarentolae promastigotes than those in T. brucei bloodstream forms.     

Isolation and immunochemical characterization of the major allergen of birch pollen (Betula verrucosa)

The classification of some of the extractable birch pollen antigens as allergens was established by crossed radioimmunoelectrophoresis (CRIE). In CRIE the major allergen (antigen 23) exhibited the strongest “radiostaining” and only a few other components of birch pollen extract were visibly radiostained. The major allergen and a preparation containing mainly the minor allergens, antigens 25 and 19, were isolated from a crude aqueous birch pollen extract by a combination of anion-exchange, size-exclusion, and chelate chromatography. Antigen 23 was purified to near homogeneity. The molecular weights and the pIs of antigens 23, 25, and 19 were determined to be 17,000 daltons, pI 5.25 (5.5, 5.0); 25,000 daltons, pI 5.0 (4.9, 5.4); and 29,000 daltons, pI 6.2 (5.4), respectively. The classification of antigen 23 as the major allergen in birch pollen was supported by results of RAST inhibition experiments, RAST screening, and skin prick testing.     

Characterization of three different human T cell membrane antigens,two being present on T lymphocyte subpopulations

Heteroantisera were raised in rabbits to thymocytes, HSB2 cells, and Sezary cells. Following absorption with Ia-positive leukemia cells, these sera appeared to be specific for different T cell antigens. Both the anti-HSB2 and the anti-Sezary sera reacted with approximately 50% and the antihymocyte serum with 100% of normal peripheral blood T lymphocytes. None of the sera reacted with B cells. The apparent molecular weights of the antigens being derected were determined by immunoprecipitation followed by SDS polyacrylamide gel eletrophoresis. A dimer of 170,000 daltons consisting of two similar 85,000-dalton polypeptide chains was immunoprecipitated by the anti-HSB2 serum whereas single polypeptides of 53,000 and 64,000 daltons were immunoprecipitated by the anti-Sezary and antithymocyte sera, respectively.     

Quantitative determination of anti-dsDNA antibodies and antibody/dsDNA stoichiometries in prepared,soluble complement-fixing antibody/dsDNA immune complexes

We have investigated quantitatively the complement-mediated binding of prepared, soluble ¹²⁵I-7S IgG antibody/³H-dsDNA immune complexes to human red blood cells (RBCs). We have performed these studies by using a detailed modification of the RBC-CF assay [Pedersen et al., J. Immun. Meth. 38, 2692–2280 (1980)] which now allows for the simultaneous measurement of both ³H-DNA and ¹²⁵I-binding to the cells. Our results indicate that, in the case of three SLE patients, their anti-dsDNA antibody titers are sufficiently high that a small fraction of their ¹²⁵I-7S IgG antibodies (ca 0.1–0.2%) can be identified as specifically anti-dsDNA. We have also used an indirect method (with ¹²⁵I-labelled rabbit anti-human IgG) for the determination of IgG anti-dsDNA antibodies in complement-fixing antibody/dsDNA immune complexes that bind to RBCs, and the results of these measurements are in reasonable agreement with the direct binding experiments. These studies have also allowed us to estimate the antibody/DNA stoichiometries in complement-fixing immune complexes. The results of these experiments may provide a useful standard for the analysis of monoclonal anti-dsDNA antibodies.     

Localization of the disulfide bridges of human histocompatibility antigens.

Both papain-solubilized and detergent-solubilized human histocompatibility antigens have been treated with NTCB (2-nitro-5-thiocyanatobenzoic acid) which cleaves these molecules at cysteine residues. A study of the fragment produced has made it possible to deduce the size and location of the two disulfide loops in these molecules. The sizes of the two loops in HLA-B7 and in the mixture HLA-B7 + 12 are about 5100 and 6600 daltons, a size similar to that of the disulfide loops found in immunoglobulins. The disulfide loops in HLA-A2 may be smaller in size. The two loops are located in middle regions of these molecules; neither the N-terminal nor the C-terminal regions contain disulfide loops.     

Interaction of immunoglobulin with actin

Actin can form specific, direct associations with immunoglobulin resulting in soluble complexes or cross-linked matrices. This interaction can be detected by four in vitro assays using purified components: (1) actin enhances the cytophilic activity of guinea pig IgG2; (2) in solutions of low ionic strength, actin and IgG2 co-precipitate: (3) soluble complexes exist in 0.1 M KCl as revealed by the displacement of actin from its expected sedimentation pattern in a gradient of sucrose when in the presence of IgG 1, IgG2, or IgM; (4) immunoglobulin (IgG1, IgG2, BGG)‡: increases the viscosity of F-actin solutions, presumably by crosslinking F-actin filaments. These data suggest that direct interaction of a cytoskeletal protein with a cell surface receptor is possible.     

MRI临床应用新进展

磁共振成像设备的飞速发展为许多高端新技术在临床的应用奠定了基础，很好地解决了临床应用过程中的伪影和心血管成像等难题，在心脏的冠脉成像、血管造影成像、弥散和灌注成像、磁共振波谱、以及功能磁共振成像等方面均取得了令人瞩目的进展，大大地满足了临床与科研的需要。     

Coupling of Metal Containing Homing Devices to Liposomes via a Maleimide Linker: Use of TCEP to Stabilize Thiol-groups without Scavenging Metals

Liposomes for drug delivery are often prepared with maleimide groups on the distal end of PEG to enable coupling of homing devices, such as antibodies, or other proteins. EDTA is used to stabilize the thiol group in the homing device for attachment to the maleimide. However, when using a homing device that contains a metal, EDTA inactivates this by scavenging of the metal. Holo-transferrin (Tf) containing two iron atoms (Fe³⁺), has a much higher affinity for the Tf receptor than apo-Tf (which does not contain any Fe³⁺). To couple Tf to a liposome, the introduction of a thiol group is necessary. During this process, by using N-succinimidyl S-acetylthioacetate (SATA), followed by 2–3 h coupling to the liposomes, Fe³⁺ is scavenged by EDTA. This causes a decreased affinity of Tf for its receptor, resulting in a decreased targeting efficiency of the liposomes.

Tris(2-carboxyethyl)phosphine (TCEP) hydrochloride is a sulfhydryl reductant that is often used in protein biochemistry. We found that TCEP (0.01 mM) does not scavenge Fe³⁺ from Tf and is able to protect thiol groups for the coupling to maleimide. Furthermore, TCEP does not interfere with the maleimide coupling itself.

In this communication, we describe the preparation of liposomes, focussing on the coupling of Tf to the maleimide linker at the distal end of PEG, without loosing Fe³⁺ from Tf. This method can be applied to other metal-containing homing devices as well.