The influence of self-MHC and non-MHC antigens on the selection of an antigen-specific T cell receptor repertoire

We have examined the influence of self-Ag on TCR expression and specificity in the immune response to the Ag pigeon cytochrome c. Previous work has shown that most Ek-restricted cytochrome c-specific T cells from B10 background mice express TCR alpha beta-heterodimers encoded by V beta 3 and V alpha 11 genes, but that T cells expressing V beta 3 proteins are eliminated due to self-tolerance in Mls-2a mouse strains. Thus, EK-restricted cytochrome c-specific T cells from Mls-2a mice fail to express any V beta 3. In the current study the influence of self-MHC and non-MHC Ag on TCR usage in the immune response to cytochrome c was further examined. First, it was demonstrated that the absence of V beta 3 expression in Mls-2a mice does not alter Ir gene function. Specifically, Mls-2a/Eb haplotype V beta 3- [C3H.SW x B10.A(5R)]F1 mice were high responders to cytochrome c despite the fact that previous structure function analyses have shown a very close correlation between Eb-restricted cytochrome c recognition and V beta 3 expression. This demonstration of the plasticity of TCR expression suggests that relatively few Ir gene defects result from tolerance induced by self-Ag. We also examined differences in V alpha 11 expression among cytochrome c-specific T cells from various H-2k haplotype mouse strains. In particular, the low level of expression of V alpha 11 in cytochrome c-specific T cells from C57BR (H-2k) mice was shown not to be due to self-tolerance. Rather, evidence for limited strain polymorphism of V alpha 11 genes, plus the fact that cytochrome c-specific T cells from F1 hybrids between H-2k, Mls-2b identical C57BR and B10.BR mice express high levels of V alpha 11, suggested the possibility that the variable V alpha 11 usage in the cytochrome c-specific responses of these two strains reflected differences in positive selection during ontogeny by non-MHC non-Mls self-Ag.     

Biosynthesis of the corrin macrocycle of coenzyme B12 in Pseudomonas denitrificans.

Studies with cell-free protein preparations from a series of recombinant strains of Pseudomonas denitrificans demonstrated that precorrin-3 is converted into a further trimethylated intermediate, named precorrin-3B, along the pathway to coenzyme B12. It was then shown that the part of the pathway from precorrin-3 (called precorrin-3A hereafter) to precorrin-6x involves three intermediates, precorrin-3B, precorrin-4, and precorrin-5. Precorrin-3B was isolated in its native (reduced) as well as its oxidized (factor-IIIB) states, and precorrin-4 was isolated in its oxidized form only (factor-IV). Both factors were in vitro precursors of precorrin-6x. The synthesis of precorrin-6x from precorrin-3A was shown to be catalyzed by four enzymes, CobG, CobJ, CobM, and CobF, intervening in this order. They were purified to homogeneity. CobG, which converts precorrin-3A to precorrin-3B, was found to be an iron-sulfur protein responsible for the oxidation known to occur between precorrin-3A and precorrin-6x, and CobJ, CobM, and CobF are the C-17, C-11, and C-1 methylases, respectively. The acetate fragment is extruded after precorrin-4 formation. This study combined with our recent structural studies on factor-IV (D. Thibaut, L. Debussche, D. Fréchet, F. Herman, M. Vuilhorgne, and F. Blanche, J. Chem. Soc. Chem. Commun. 1993:513-515, 1993) and precorrin-3B (L. Debussche, D. Thibaut, M. Danzer, F. Debu, D. Fréchet, F. Herman, F. Blanche, and M. Vuilhorgne, J. Chem. Soc. Chem. Commun. 1993:1100-1103, 1993) provides a first step-by-step picture of the sequence of the enzymatic reactions leading to the corrin ring in P. denitrificans.     

A bifunctional protein from Pseudomonas denitrificans carries cobinamide kinase and cobinamide phosphate guanylyltransferase activities.

The two consecutive activities of the cobalamin biosynthetic pathway that catalyze the conversion of cobinamide to cobinamide phosphate (cobinamide kinase) and of cobinamide phosphate to GDP-cobinamide (cobinamide phosphate guanylytransferase) were shown to be carried by the same protein in Pseudomonas denitrificans. This bifunctional protein was purified to homogeneity by high-performance liquid chromatography of extracts of a recombinant strain of this microorganism, and the sequence of the first 10 amino acid residues at the N terminus was determined. Both activities were specific to the coenzyme forms of the corrinoid substrates and exhibited an optimum pH at 8.8. Both ATP and GTP were shown to be in vitro gamma-phosphate donors for cobinamide kinase. However, competition experiments demonstrated that ATP was the preferred substrate, a result that can be explained in terms of the kinetic properties of the enzyme. Labeling experiments established that the phosphate group of cobinamide phosphate is quantitatively retained as the inner phosphate of GDP-cobinamide during the guanylyltransferase reaction. The native protein had an apparent molecular weight of 40,000, as estimated by gel filtration, and consisted of two identical subunits of Mr 20,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein had an isoelectric point of 5.35 and contained a high-affinity GTP-binding site (Kaff.(GTP) = 0.22 microM). Binding of GTP onto this site resulted in a marked increase of the affinity of cobinamide kinase for cobinamide. This property and other kinetic properties may regulate the enzyme and prevent the accumulation of cobinamide phosphate.     

The final step in the biosynthesis of hydrogenobyrinic acid is catalyzed by the cobH gene product with precorrin-8x as the substrate.

The final enzymatic reaction in the conversion of precorrin-6x to hydrogenobyrinic acid by cell-free protein preparations from Pseudomonas denitrificans was shown to be inhibited by hydrogenobyrinic acid. Use was made of this property to prepare the last biosynthetic precursor of hydrogenobyrinic acid, named precorrin-8x. Double-labeling experiments, mass spectrometry, and UV-visible light spectroscopy studies established that precorrin-8x was at the oxidation level of a corrin and differed from precorrin-6x by two additional methyl groups (presumably at C-5 and C-15) and decarboxylation of the acetic acid side chain at C-12. Precorrin-8x was not a corrin but had the same mass as hydrogenobyrinic acid, thus showing that this latter compound is synthesized from the former by a rearrangement. The enzyme catalyzing this rearrangement was purified 80-fold to homogeneity from a recombinant strain of P. denitrificans, sequenced at its N terminus, and shown to be encoded by the cobH gene. It was identical to the previously described hydrogenobyrinic acid-binding protein (F. Blanche, D. Thibaut, D. Frechet, M. Vuilhorgne, J. Crouzet, B. Cameron, G. Müller, K. Hlineny, U. Traub-Eberhard, and M. Zboron, Angew. Chem. Int. Ed. Engl. 29:884-886, 1990). This enzyme had a Km of 0.91 +/- 0.04 microM and a Vmax of 230 nmol h-1 mg-1 at pH 7.7 and was competitively inhibited by hydrogenobyrinic acid with a Ki of 0.17 +/- 0.01 microM. It is proposed that the cobH gene product is a mutase which transfers the methyl group from C-11 to C-12.     

Compartmental models with Erlang distributed residence times and random rate coefficients

In this paper a stochastic model for a two-compartment system which incorporates Erlang residence time distributions (i.e. the residence times have the gamma distribution where the shape parameters assume integer values only) into each compartment is generalized to include random rate coefficients. Analytical forms of the model are derived for the case where the rate coefficients have gamma densities. A relationship is established between the new models and existing models that are in current practical usage.     

Purification and characterization of cobyrinic acid a,c-diamide synthase from Pseudomonas denitrificans.

Cobyrinic acid a,c-diamide synthase, which catalyzes the conversion of cobyrinic acid to cobyrinic acid a,c-diamide via the intermediate formation of cobyrinic acid c-monoamide, was purified 155-fold to homogeneity from extracts of a recombinant strain of Pseudomonas denitrificans by high-performance liquid chromatography. The enzyme has an apparent molecular weight of 86,000 and consists of two identical subunits of Mr 45,000, as estimated by gel electrophoresis under denaturing conditions. Stepwise Edman degradation provided the N-terminal sequence of the first 15 amino acids. Glutamine was shown to be the preferred amino group donor (Km = 20.3 microM), but it could be replaced by ammonia (Km = 12 mM). The reaction was ATP dependent and exhibited a broad optimum pH around 7.3. Km values for (CN,aq)cobyrinic acid, (aq)2cobyrinic acid, and (CN,aq)cobyrinic acid c-monoamide were 160, greater than or equal to 250, and 71 microM, respectively. Hydrogenobyrinic acid and hydrogenobyrinic acid c-monoamide were shown to be much better substrates, with Km values of 0.41 and 0.21 microM, respectively.     

Value of the specific distinction between Ascaris lumbricoïdes linné 1758 and Ascaris suum Goeze 1782

Ansel M. and Thibaut M. 1973. Value of the specific distinction between Ascaris lumbricoïdes Linné 1758 and Ascaris suum Goeze 1782. International Journal for Parasitology3: 317–319. Study by scanning electron microscopy of heads of Ascaris lumbricoides Linné 1758 and Ascaris suum Goeze 1782 showed typical distinctive characteristics between these two species. There are differences in the appearance of the rows of denticules and the shapes of the lips.     

An Investigation of Freezing of Supercooled Water on Anti-Freeze Protein Modified Surfaces

This work investigates how functionalization of aluminium surfaces with natural type III Anti-Freeze Protein (AFP) affects the mechanism of heterogeneous ice nucleation. First the bulk ice nucleation properties of distilled water and aqueous solution of AFP were evaluated by differential scanning calorimetry. Then the modified surface was characterized by Secondary Ions Mass Spectroscopy (SIMS), Fourier Transform InfraRed (FTIR) spectroscopy and contact angle measurement. Freezing experiments were then conducted in which water droplets underwent a slow controlled cooling. This study shows that compared to uncoated aluminium, the anti-freeze proteins functionalized surfaces exhibit a higher and narrower range of freezing temperature. It was found that these proteins that keep living organisms from freezing in cold environment act in the opposite way once immobilized on surfaces by promoting ice nucleation. Some suggestions regarding the mechanism of action of the observed phenomena were proposed based on the Classical Nucleation Theory (CNT).