Identification of form III conformers in tRNAPhe from Escherichia coli by intramolecular photo-cross-linking

In the absence of divalent cations, at neutral pH, low ionic strength, and low to moderate temperature, tRNAs are known to be in a denatured form, designated form III in the tRNA phase diagram by Cole et al. [Cole, P. E., Yang, S. R., & Crothers, D. M. (1972) Biochemistry 11, 4358-4368]. Form III tRNAPhe from Escherichia coli has been studied at pH 7, 5 mM Na+, and 10 degrees C. As judged from ethidium bromide intercalation, it exhibits extensive secondary structure. 4-Thiouridine in position 8 of the tRNAPhe sequence was used as a built-in photoaffinity probe. Spectroscopic and spectrofluorometric analysis in the near-UV range of form III tRNAPhe irradiated with broad-band near-UV light to completion of the reaction before or after reduction with NaBH4 revealed that the Pdo(4-5)Cyt (8-C) and Pdo(4-5)Urd (8-U) adducts form in equimolar yield. In different experiments, the overall yield of s4U conversion to these adducts varies between 20 and 40%. The remaining s4U is photolyzed to weakly absorbing product(s) in the near-UV range. The disappearance of s4U follows biexponential kinetics while the 8-C adduct formation follows monoexponential kinetics, indicating the presence of at least two tRNA classes of conformers, not in equilibrium on the time scale of the reaction. Migration on a denaturing polyacrylamide gel of irradiated form III tRNAPhe revealed three main bands, D1, D2, and D3, and no slowly migrating tRNA dimers. D1 migrates at the control position and presumably contains the photolysis product(s) P. The fast-migrating D2 and D3 bands contain 8-Pyr cross-links which were identified by sequence analysis as 8-(66-68) in D2 and 8-(40-43) and 8-(59-62) in D3. On the basis of these data, it is proposed that the minor poorly photoreactive class II conformers are the cloverleaf and close variants, whereas the major class I cross-linkable conformers are essentially long-extended secondary structures. Clearly, our data demonstrate the polymorphism of form III tRNAPhe.     

KLH-specific,I-E/C-restricted clones of proliferating T lymphocytes

The recognition of keyhole limpet hemocyanin by a substantial proportion of proliferating clones of murine T lymphocytes was found to be restricted by the I-E/C^k molecule, which is a combinatorial product of genes located in the I-A (Ae) and I-EIC (E) subregions of the murine major histocompatibility complex. The respective roles of the Ae (polymorphic) and E (oligomorphic) gene products in the expression of the structures which are used as restriction elements by these T-cell clones was analyzed by mating parental strains unable to present the antigen and bearing selected Ae and E alleles. Efficient complementation for antigen presentation was found to require the expression by accessory cells of the Ae^k-gene product, whereas all E allelic molecules were functionally equivalent. These results (a) indicate that the immunoregulatory role of I-region gene products, initially described for molecules selected for their limited number of antigenic epitopes, also applies to complex multiepitopic antigens; (b) illustrate the advantage which results from the diversity of the la molecules expressed by accessory cells for the development of potent immune responses; and (c) suggest that a correlation might exist between the degree of polymorphism of a given family of H-2 allelic molecules and their ability to be used as restriction elements for antigen recognition by T lymphocytes.Abbreviations used in this paper FCS fetal calf serum - Hepes N-2-hydroxypiperazine-N-2 ethanesulfonic acid; in polypeptides - G glutamate - L lysine - Ø phenylalanine - KLH keyhole limpet hemocyanin - m. Ab. monoclonal antibodies - T cells thymus-derived cells     

Directed molecular evolution of antibodies

Antibodies play a key role in the immune system, are characterized by a homogeneous overall structure and by their ability to interact with an almost unlimited number of compounds. Encoded by a fixed number of genes, they acquire their specificity and affinity of recognition after a succession of genetic recombination and molecular mutation processes. Since the pioneer works of Kohler and Milstein in 1975 describing the possibility of producing monoclonal antibodies with pre-determined specificity, the use of antibodies in the fields of research, diagnosis and therapy has never stopped increasing. Thus, about twenty monoclonal antibodies have yet been authorized to be used in human immunotherapy. However, a majority of these molecules have been engineered to bring them into line with their clinical use: chimerization, humanization, recombinant expression of single or fused fragments. Furthermore, the recent development of in vitro molecular evolution approaches now make it possible to engineer the affinity, the specificity as well as the stability of monoclonal antibodies. The potential of in vitro molecular evolution of antibodies will be illustrated through the example of the specificity improvement of an anti-progesterone antibody.     

Synthesis of 3'-O2-(azaheterocycle)-thymidines

The synthesis of 3'-O2-(azaheterocycle)-thymidines is presented from 1-thia-3-aza- 1,3-butadiene precursors (N-thioacylamidines). A variety of heterocycles is accessible using the dienic, the electrophilic or the nucleophilic reactivity of these thia-azabutadiene systems. 3'-O2-(azaheterocycle)-thymidine analogues are regarded as potential substrates to interfere with the DNA-polymerization process.     

Oligomerization of Escherichia coli enterotoxin b through its C-terminal hydrophobic alpha-helix

Using a chemical cross-linker and gel electrophoresis or a dot blot overlay assay, we studied protein-protein interaction of STb toxin, a 48-residue amphiphilic polypeptide causing intestinal disorders. For the first time, we report on the oligomerization property of STb. This enterotoxin forms hexamers and heptamers in a temperature-independent fashion in presence or absence of its receptor (sulfatide) anchored in a 50-nm liposome or as a free molecule. Full STb structure integrity is necessary for its oligomerization as this process is not observed under reducing conditions in the presence of beta-mercaptoethanol. STb treatment with tetramethylurea (TMU) and different detergents prevented oligomerization. Site-directed mutagenesis decreasing overall STb hydrophobicity in the hydrophobic alpha-helix resulted in the incapacity to form oligomers. Taken together, these data suggest that the C-terminal hydrophobic alpha-helix corresponds to the domain of STb-STb inter-binding where hydrophobic interaction is involved.     

Antigenic differences among Campylobacter fetus S-layer proteins.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of S-layer proteins extracted from Campylobacter fetus strains by using acid glycine buffer showed that the predominant S-layer proteins of different strains had subunit molecular weights in the range of 90,000 to 140,000. Electron microscopy revealed oblique S-layer lattices with a spacing of approximately 5.6 nm (gamma = 75 degrees) on wild-type strains VC1, VC119, VC202, and VC203. Three variants of C. fetus VC119 producing a predominant S-layer subunit protein of different molecular weight (Mr) from that of the parent were also examined. Each variant produced an oblique lattice morphologically indistinguishable from that of the parent. Amino-terminal sequence analysis showed that the S-layer proteins of the VC119 parent and variants were identical up to residue 18 and that this sequence differed from but was related to the first 16 N-terminal residues shared by the S-layer proteins of the three other wild-type C. fetus isolates. Western immunoblot analysis with an antiserum prepared to the VC119 protein and an antiserum prepared to C. fetus 84-40 LP (Z. Pei, R. T. Ellison, R. V. Lewis, and M. J. Blaser, J. Biol. Chem. 263:6416-6420, 1988) showed that strains of C. fetus were capable of producing S-layer proteins with at least four different antigenic specificities. Immunoelectron microscopy with antiserum to the VC119 S-layer protein showed that C. fetus cultures contained cells with immunoreactive oblique S-layer lattices as well as cells with oblique S-layer lattices which did not bind antibody. This suggests that C. fetus S-layer proteins undergo antigenic variation. Thermal denaturation experiments indicated that the antigenicity conferred by the surface-exposed C. fetus S-layer epitopes was unusually resistant to heat, and the thermal stability appeared to be due to the highly organized lattice structure of the S. layer. Protease digestion of purified VC119 S-layer protein revealed a trypsin-, chymotrypsin-, and endoproteinase Glu-C-resistant domain with an apparent Mr of 110,000, which carried the majority of the epitopes of the S-layer protein, and a small enzyme-sensitive domain. The trypsin- and chymotrypsin-resistant polypeptides shared an overlapping sequence which differed from the N-terminal sequence of the intact S-layer protein.     

Energy density of anchovy Engraulis encrasicolus in the Bay of Biscay

The energy density ( E _D) of anchovy Engraulis encrasicolus in the Bay of Biscay was determined by direct calorimetry and its evolution with size, age and season was investigated. The water content and energy density varied seasonally following opposite trends. The E _D g⁻¹ of wet mass ( M _W) was highest at the end of the feeding season (autumn: c . 8 kJ g⁻¹ M _W) and lowest in late winter ( c . 6 kJ g⁻¹ M _W). In winter, the fish lost mass, which was partially replaced by water, and the energy density decreased. These variations in water content and organic matter content may have implications on the buoyancy of the fish. The water content was the major driver of the energy density variations for a M _W basis. A significant linear relationship was established between E _D g⁻¹ ( y ) and the per cent dry mass ( M _D; x ): y =−4·937 + 0·411 x . In the light of the current literature, this relationship seemed to be not only species specific but also ecosystem specific. Calibration and validation of fish bioenergetics models require energy content measurements on fish samples collected at sea. The present study provides a first reference for the energetics of E. encrasicolus in the Bay of Biscay.     

Comparative studies of D-myoinositol 1,4,5-trisphosphate and synthetic 6-deoxy D-myoinositol 1,4,5-trisphosphate: binding and calcium release activity in rat parotid microsomes.

In this study, we report our data on the binding of D-myoinositol(1,4,5)P3 and of 6-deoxy D-myoinositol(1,4,5)P3 to a rat parotid microsomal fraction and their effect on Ca2+ release. The binding affinity and the potency of 6-deoxy Ins(1,4,5)P3 to induce Ca2+ release are about 100 times lower than those of Ins(1,4,5)P3. However, maximal concentrations of both inositol trisphosphates induce similar calcium efflux and present comparable displacement of radioligand binding. Experiments were performed to exclude that the microsomal preparations used display rapid metabolism of Ins(1,4,5)P3 or 6-deoxy Ins(1,4,5)P3 during binding and Ca2+ release. We also report that, in permeabilized rat parotid acini preparations, 6-deoxy Ins(1,4,5)P3 is about 100 times less potent than Ins(1,4,5)P3 in inducing Ca2+ release. These data indicate that removal of the hydroxyl group in position 6 of the Ins(1,4,5)P3 molecule severely reduces its binding affinity which seems, in a large part at least, responsible for the reported loss of potency in mobilizing Ca2+. Nevertheless, 6-deoxy Ins(1,4,5)P3 seems to be a full agonist for the release of Ca2+.