Knowledge-based design of reagentless fluorescent biosensors from recombinant antibodies

The possibility of obtaining from any antibody a fluorescent conjugate which responds to the binding of the antigen by a variation of its fluorescence, would be of great interest in the analytical sciences and for the construction of protein chips. This possibility was explored with antibody mAbD1.3 directed against hen egg white lysozyme. Rules of design were developed to identify the residues of the antibody to which a fluorophore could be chemically coupled, after changing them to cysteine by mutagenesis. These rules were based on: the target residue belonging to a topological neighbourhood of the antigen in the structure of the complex between antibody and antigen; its absence of functional importance for the interaction with the antigen; and its solvent accessibility in the structure of the free antibody. Seventeen conjugates between the single-chain variable fragment scFv of mAbD1.3 and an environment-sensitive fluorophore were constructed. For six of the ten residues which fully satisfied the design rules, the relative variation of the fluorescence intensity between the free and bound states of the conjugate was comprised between 12 and 75% (in non-optimal buffer), and the affinity of the conjugate for lysozyme remained unchanged relative to the parental scFv. In contrast, such results were true for only one of the seven residues which failed to satisfy one of the rules and were used as controls. One of the conjugates was studied in more detail. Its fluorescence increased proportionally to the concentration of lysozyme in a nanomolar range, up to 90% in a defined buffer, and 40% in serum. This increase was specific for hen egg lysozyme and it was not observed with a closely related protein, turkey egg lysozyme. The residues which gave operational conjugates (six in V(L) and one in V(H)), were located in the immediate vicinity of residues which are functionally important, along the sequence of FvD1.3. The results suggest rules of design for constructing antigen-sensitive fluorescent conjugates from any antibody, in the absence of structural data.     

Isolation of mannose 6-phosphate reductase cDNA,changes in enzyme activity and mannitol content in broomrape (Orobanche ramosa) parasitic on tomato roots

We are interested in developing a control strategy efficient at the early stages of subterranean development of Orobanche in the inhibition of mannose 6-phosphate reductase (M6PR, EC 1.1.1.224), the key enzyme of mannitol production in the parasite. We examined M6PR gene expression during pre-conditioning, germination, procaulome growth, underground shoot development and emergence of Orobanche ramosa L. attached to tomato roots, the enzyme activity at each of the above stages and the level of stored mannitol in the parasite. A 1120-pb length cDNA isolated by 3' and 5'RACE was identified as a M6PR sequence by cDNA expression in E. coli and M6PR activity measurement. Only one M6PR gene was detected in O. ramosa following southern blot analysis. M6PR expression, analysed by RT-PCR, was constant from the pre-conditioned seed to the emergence of broomrape, i.e. M6PR expression is constitutive in Orobanche . M6PR activity was also detected in pre-conditioned seeds and attachment to tomato roots resulted in a two-fold increase in enzyme activity during tubercle enlargement and crown root formation. Hexose and mannitol accumulation was strongly enhanced in the attached parasite, with accumulation primarily in the shoot. These results support the prospect of utilizing M6PR inhibitors as early applied herbicides to control this parasite in the early stages of its development.     

Study of the cytotoxic effect of a peptidic inhibitor of the p53-hdm2 interaction in tumor cells

Inhibitors of the p53-hdm2 interaction are attractive molecules for stimulating the p53 pathway in tumors. In this report, an inhibitor of the p53-hdm2 interaction, the AP peptide, is used to activate p53 in tumor cells expressing various levels of hdm2 protein. It induces apoptosis only in cells expressing high endogenous levels of hdm2 protein. The absence of apoptosis in tumor cells with low hdm2 levels is due not to alterations in the p53-dependent apoptotic pathway but to a different regulation of this pathway. The peptide is also less toxic for non-tumor cells than for tumor cells overexpressing the hdm2 protein.     

Beta(1)/beta(2)/beta(3)-adrenoceptor knockout mice are obese and cold-sensitive but have normal lipolytic responses to fasting

Catecholamines are viewed as major stimulants of diet- and cold-induced thermogenesis and of fasting-induced lipolysis, through the β-adrenoceptors (β₁/β₂/β₃). To test this hypothesis, we generated β₁/β₂/β₃-adrenoceptor triple knockout (TKO) mice and compared them to wild type animals. TKO mice exhibited normophagic obesity and cold-intolerance. Their brown fat had impaired morphology and lacked responses to cold of uncoupling protein-1 expression. In contrast, TKO mice had higher circulating levels of free fatty acids and glycerol at basal and fasted states, suggesting enhanced lipolysis. Hence, β-adrenergic signalling is essential for the resistance to obesity and cold, but not for the lipolytic response to fasting.     

Role of prolyl hydroxylation in oncogenically stabilized hypoxia-inducible factor-1alpha

Stabilization of the hypoxia-inducible factor-1 (HIF-1) protein is essential for its role as a regulator of gene expression under low oxygen conditions. Here, employing a novel hydroxylation-specific antibody, we directly show that proline 564 of HIF-1alpha and proline 531 of HIF-2alpha are hydroxylated under normoxia. Importantly, HIF-1alpha Pro-564 and HIF-2alpha Pro-531 hydroxylation is diminished with the treatment of hypoxia, cobalt chloride, desferrioxamine, or dimethyloxalyglycine, regardless of the E3 ubiquitin ligase activity of the von Hippel-Lindau (VHL) tumor suppressor gene. Furthermore, in VHL-deficient cells, HIF-1alpha Pro-564 and HIF-2alpha Pro-531 had detectable amounts of hydroxylation following transition to hypoxia, indicating that the post-translational modification is not reversible. The introduction of v-Src or RasV12 oncogenes resulted in the stabilization of normoxic HIF-1alpha and the loss of hydroxylated Pro-564, demonstrating that oncogene-induced stabilization of HIF-1alpha is signaled through the inhibition of prolyl hydroxylation. Conversely, a constitutively active Akt oncogene stabilized HIF-1alpha under normoxia independently of prolyl hydroxylation, suggesting an alternative mechanism for HIF-1alpha stabilization. Thus, these results indicate distinct pathways for HIF-1alpha stabilization by different oncogenes. More importantly, these findings link oncogenesis with normoxic HIF-1alpha expression through prolyl hydroxylation.     

Localization of the yeast RNA polymerase I-specific subunits

The spatial distribution of four subunits specifically associated to the yeast DNA-dependent RNA polymerase I (RNA pol I) was studied by electron microscopy. A structural model of the native enzyme was determined by cryo-electron microscopy from isolated molecules and was compared with the atomic structure of RNA pol II Delta 4/7, which lacks the specific polypeptides. The two models were aligned and a difference map revealed four additional protein densities present in RNA pol I, which were characterized by immunolabelling. A protruding protein density named stalk was found to contain the RNA pol I-specific subunits A43 and A14. The docking with the atomic structure showed that the stalk protruded from the structure at the same site as the C-terminal domain (CTD) of the largest subunit of RNA pol II. Subunit A49 was placed on top of the clamp whereas subunit A34.5 bound at the entrance of the DNA binding cleft, where it could contact the downstream DNA. The location of the RNA pol I-specific subunits is correlated with their biological activity.