Directional electron transfer in ruthenium-modified horse heart cytochrome c

Cytochrome c can be modified by [(NH3)5RuII/III-] specifically at the imidazole moiety of histidine 33, and we have recently discussed the thermodynamics and kinetics of electron transfer within this modified protein. X-ray crystal structures of the oxidized and reduced forms of tuna cytochrome c indicate that the separation between the haem group of cytochrome c and the ruthenium label is 12-16 A. Internal electron transfer from the [(NH3)5RuII-] centre to the Fe(III) haem centre occurs with a rate constant k congruent to 53 s-1 (25 degrees C) (delta H = 3.5 kcal mol-1, delta S = -39 EU), as measured by pulse radiolysis. The measured unimolecular rate constant, k congruent to 53 s-1, is on the same timescale as a number of conformational changes that occur within the cytochrome c molecule. These results raise the question of whether electron transfer or protein conformational change is the rate limiting step in this process. We describe here an experiment that probes this intramolecular electron transfer step further. It involves reversing the direction of electron transfer by changing the redox potential of the ruthenium label. Electron transfer in the new ruthenium-cytochrome c derivative described here is from haem(II) to the Ru(III) label, whereas in (NH3)5Ru-cytochrome c the electron transfer is from Ru(II) to haem(III). Intramolecular electron transfer from haem(II) to Ru(III) in the new ruthenium-cytochrome c described here proceeds much slower (greater than 10(5) times) than the electron transfer from Ru(II) to haem(III) in the (NH3)5Ru-cytochrome c. We therefore conclude that electron transfer in cytochrome c is directional, with the protein envelope presumably involved in this directionality.     

Structure of pre-pro-von Willebrand factor and its expression in heterologous cells

Von Willebrand factor (vWF), a multifunctional haemostatic glycoprotein derived from endothelial cells and megakaryocytes, mediates platelet adhesion to injured subendothelium and binds coagulation factor VIII in the circulation. Native vWF is a disulphide-bonded homopolymer; the monomeric subunits, of apparent relative molecular mass (Mr) 220,000 (220K) are derived from an intracellular precursor estimated at 260-275K. Multimer assembly is preceded by the formation of dimers, linked near their C-termini, which then assemble into filamentous polymers. The importance of the removal of the large vWF pro-polypeptide during multimer assembly, and whether this or other stages of the complex post-translational processing require components specific to endothelial cells or megakaryocytes, is unknown. Here we report an analysis of the complete sequence of pre-pro-vWF and expression of the molecule in heterologous cells. The vWF precursor is composed of several repeated subdomains. When expressed in COS and CHO cells, it is cleaved and assembled into biologically active high relative molecular mass disulphide bonded multimers. This suggests that the information for assembly of this complex molecule resides largely within its primary structure.     

Implications of fragile X expression in normal males for the nature of the mutation

The fragile site at Xq27, associated with a common form of X-linked mental retardation (XLMR), is expressed in a variable proportion of the peripheral lymphocytes of affected males when the cells are cultured under thymidylate stress (Td stress) produced by folate or thymidylate deprivation. Some clinically normal males--transmitting males--are known to carry and transmit the fragile X mutation and yet show no cytogenetic expression in lymphocytes. Normal males with no family history of X-linked mental retardation express the site only rarely. When the fragile X chromosome from affected males is isolated in a rodent genetic background by somatic cell hybridization, the level of expression is similar to that seen in lymphocytes under Td stress. Here we show that X chromosomes from two transmitting males and two normal control males, all of which were fragile X negative in lymphocytes or lymphoblasts, could be made to express the fragile site in hybrids, although at levels that were below those seen in hybrids from affected males. Furthermore, transmitting males could be differentiated from normal males by their significantly higher expression rates when hybrids were exposed to caffeine before cytogenetic harvest. One male chimpanzee also showed low level expression in hybrid cells. These data suggest that the hybrid system lowers the threshold for fragile X expression, a fragile site at Xq27 may be present on all human and chimpanzee X chromosomes and constitutes a previously unrecognized common fragile site and the hybrid system with caffeine post-treatment can distinguish between the common Xq27 fragile site of control males, the occult mutant fragile site of a transmitting male, and the fully expressed fragile site of an affected male with XLMR. Thus the mutation producing XLMR may represent a multi-step alteration of a naturally occurring DNA sequence producing a continuum of cytogenetic expression and a threshold for clinical manifestation.     

SB-restricted presentation of influenza and herpes simplex virus antigens to human T-lymphocyte clones

The HLA-D region of the human major histocompatibility complex (MHC) has been shown to be homologous to the murine I region in terms of both structure and function. Both regions encode class II MHC molecules which restrict T-lymphocyte interactions with antigen-presenting cells. We have recently described the MHC restriction and antigen specificities of human T-lymphocyte clones directed at strain A influenza virus. The majority of T-lymphocyte clones recognized antigen in the context of cell surface interaction products encoded by HLA-D/DR genes. However, a few clones recognized antigen presented by cells histoincompatible for D/DR antigens. We report here that some of these clones recognized viral antigens in association with antigens encoded by genes identical with or closely linked to the recently described secondary B-cell (SB) locus of the MHC. This is the first report that SB-restricted antigen recognition may form an integral part of normal, human immune responses.     

Bone marrow genotoxicity of N-methyl, N-nitrosourea (NMU): n-alkanols as sister chromatid exchange (SCE) anti-inducers

The in vivo SCE test was used to demonstrate significant inhibition of NMU bone marrow genotoxicity by pretreatment of Chinese hamsters with n-alkanols. Our findings exclude a loss of intracellular DNA alkylation potential through a competitive direct reaction of NMU with the weakly nucleophilic polar end of the n-alkanols, but not through methylations of nucleophilic membrane sites possibly liberated by structural modifications which the membrane-active amphiphilics induce.     

Steady-state kinetic studies with the polysulfonate U-9843, an HIV reverse transcriptase inhibitor

The tetramer of ethylenesulfonic acid (U-9843) is a potent inhibitor of HIV-1 RT^* and possesses excellent antiviral activity at nontoxic doses in HIV-1 infected lymphocytes grown in tissue culture. Kinetic studies of the HIV-1 RT-catalyzed RNA-directed DNA polymerase activity were carried out in order to determine if the inhibitor interacts with the template: primer or the deoxyribonucleotide triphosphate (dNTP) binding sites of the polymerase. Michaelis-Menten kinetics, which are based on the establishment of a rapid equilibrium between the enzyme and its substrates, proved inadequate for the analysis of the experimental data. The data were thus analyzed using steady-state Briggs-Haldane kinetics assuming that the template:primer binds to the enzyme first, followed by the binding of the dNTP and that the polymerase is a processive enzyme. Based on these assumptions, a velocity equation was derived which allows the calculation of all the specific forward and backward rate constants for the reactions occurring between the enzyme, its substrates and the inhibitor. The calculated rate constants are in agreement with this model and the results indicated that U-9843 acts as a noncompetitive inhibitor with respect to both the template:primer and dNTP binding sites. Hence, U-9843 exhibits the same binding affinity for the free enzyme as for the enzyme-substrate complexes and must inhibit the RT polymerase by interacting with a site distinct from the substrate binding sites. Thus, U-9843 appears to impair an event occurring after the formation of the enzyme-substrate complexes, which involves either an event leading up to the formation of the phosphoester bond, the formation of the ester bond itself or translocation of the enzyme relative to its template:primer following the formation of the ester bond.     

Age-related lipid peroxidation in the digestive gland of mussels: The role of the antioxidant defence systems

Summary The main cellular defence systems against free radical-mediated oxidative stress are significantly reduced in the dige⁺ive gland of aged (>10 years old) compared to younger (2–4 years old) mussels (Mytilus edulis L.). Moreover, the concentration of lipid peroxidation products (malondialdehyde) is increased in the same age group with respect to younger animals. The obtained data indicate that an impairment of the antioxidant defence systems would render the older animals more susceptible to peroxidative stress, thus supporting the general significance of the free radical theory of aging.