Activation of mammalian tyrosinase by ferrous ions

Kinetic experiments are reported showing that mammalian tyrosinase from B16 mouse melanoma is significantly activated by catalytic amounts of ferrous ions. Monitoring of tyrosine oxidation by both dopachrome formation and oxygen consumption showed that ferrous ions at micromolar concentrations induce a marked enzymatic activity with 0.01 U/ml of highly purified tyrosinase, whereas no detectable reaction occurs in the absence of metal over a sufficiently prolonged period of time. The extent of the activating effect, which is specific for the reduced form of iron, is proportional to the concentration of the added metal with a typical saturation profile, no further effect being observed beyond a threshold value. Changing the buffer system from phosphate to hepes or tris results in a marked decrease of the Fe2(+)-induced activation. Scavengers of active oxygen species, such as superoxide dismutase, catalase, formate and mannitol have no detectable effect on the tyrosinase activity. These results are accounted for in terms of an activation mechanism involving reduction of the cupric ions at the active site of the resting enzyme.     

Trichostatin A improves histone acetylation in bovine somatic cell nuclear transfer early embryos

Epigenetic aberrancies likely preclude correct and complete nuclear reprogramming following somatic cell nuclear transfer (SCNT), and may underlie the observed reduced viability of cloned embryos. In the present study, we tested the effects of the histone deacetylase inhibitor (HDACi), trichostatin A (TSA), on development and histone acetylation of cloned bovine preimplantation embryos. Our results indicated that treating activated reconstructed SCNT embryos with 50 nM TSA for 13 h produced eight-cell embryos with levels of acetylation of histone H4 at lysine 5 (AcH4K5) similar to fertilized counterparts and significantly greater than in control NT embryos (p < 0.005). Further, TSA treatment resulted in SCNT embryos with preimplantation developmental potential similar to fertilized counterparts, as no difference was observed in cleavage and blastocyst rates or in blastocyst total cell number (p > 0.05). Measurement of eight selected developmentally important genes in single blastocysts showed a similar expression profile among the three treatment groups, with the exception of Nanog, Cdx2, and DNMT3b, whose expression levels were higher in TSA-treated NT than in in vitro fertilized (IVF) embryos. Data presented herein demonstrate that TSA can improve at least one epigenetic mark in early cloned bovine embryos. However, evaluation of development to full-term is necessary to ascertain whether this effect reflects a true increase in developmental potential.     

Regulatory mechanisms differ in UMP kinases from gram-negative and gram-positive bacteria

In this work, we examined the regulation by GTP and UTP of the UMP kinases from eight bacterial species. The enzyme from Gram-positive organisms exhibited cooperative kinetics with ATP as substrate. GTP decreased this cooperativity and increased the affinity for ATP. UTP had the opposite effect, as it decreased the enzyme affinity for ATP. The nucleotide analogs 5-bromo-UTP and 5-iodo-UTP were 5-10 times stronger inhibitors than the parent compound. On the other hand, UMP kinases from the Gram-negative organisms did not show cooperativity in substrate binding and catalysis. Activation by GTP resulted mainly from the reversal of inhibition caused by excess UMP, and inhibition by UTP was accompanied by a strong increase in the apparent K(m) for UMP. Altogether, these results indicate that, depending on the bacteria considered, GTP and UTP interact with different enzyme recognition sites. In Gram-positive bacteria, GTP and UTP bind to a single site or largely overlapping sites, shifting the T R equilibrium to either the R or T form, a scenario corresponding to almost all regulatory proteins, commonly called K systems. In Gram-negative organisms, the GTP-binding site corresponds to the unique allosteric site of the Gram-positive bacteria. In contrast, UTP interacts cooperatively with a site that overlaps the catalytic center, i.e. the UMP-binding site and part of the ATP-binding site. These characteristics make UTP an original regulator of UMP kinases from Gram-negative organisms, beyond the common scheme of allosteric control.     

Application of a simple likelihood ratio approximant to protein sequence classification

MOTIVATION: Likelihood ratio approximants (LRA) have been widely used for model comparison in statistics. The present study was undertaken in order to explore their utility as a scoring (ranking) function in the classification of protein sequences. RESULTS: We used a simple LRA-based on the maximal similarity (or minimal distance) scores of the two top ranking sequence classes. The scoring methods (Smith-Waterman, BLAST, local alignment kernel and compression based distances) were compared on datasets designed to test sequence similarities between proteins distantly related in terms of structure or evolution. It was found that LRA-based scoring can significantly outperform simple scoring methods.     

Effect of metal ions on the rearrangement of dopachrome

In vitro experiments are reported showing that a number of transition metal ions exert a profound influence on both the kinetics and chemical course of the rearrangement of dopachrome, a key step in the biosynthesis of melanins. HPLC analysis shows that Cu2+, Ni2+ and Co2+ are particularly effective in inducing the non-decarboxylative rearrangement of dopachrome at physiological pH values, leading mainly to 5,6-dihydroxyindole-2-carboxylic acid, whereas in the absence of metal ions the reaction proceeds with concomitant loss of carbon dioxide to give almost exclusively 5,6-dihydroxyindole. Kinetic experiments provide evidence that the rate of the metal-promoted rearrangement is first order with respect to both aminochrome and metal concentration and decreases in the presence of increasing concentrations of EDTA, consistent with a mechanism involving a direct 1:1 dopachrome-metal ion interaction in the transition state. When considered in the light of the known metal accumulation in pigmented tissues, the results of this study provide a new entry into the regulatory mechanisms involved in the biosynthesis of melanins.     

Vaginal Immunization to Elicit Primary T-Cell Activation and Dissemination

Primary T-cell activation at mucosal sites is of utmost importance for the development of vaccination strategies. T-cell priming after vaginal immunization, with ovalbumin and CpG oligodeoxynucleotide adjuvant as model vaccine formulation, was studied in vivo in hormone-synchronized mice and compared to the one induced by the nasal route. Twenty-four hours after both vaginal or nasal immunization, antigen-loaded dendritic cells were detected within the respective draining lymph nodes. Vaginal immunization elicited a strong recruitment of antigen-specific CD4⁺ T cells into draining lymph nodes that was more rapid than the one observed following nasal immunization. T-cell clonal expansion was first detected in iliac lymph nodes, draining the genital tract, and proliferated T cells disseminated towards distal lymph nodes and spleen similarly to what observed following nasal immunization. T cells were indeed activated by the antigen encounter and acquired homing molecules essential to disseminate towards distal lymphoid organs as confirmed by the modulation of CD45RB, CD69, CD44 and CD62L marker expression. A multi-type Galton Watson branching process, previously used for in vitro analysis of T-cell proliferation, was applied to model in vivo CFSE proliferation data in draining lymph nodes 57 hours following immunization, in order to calculate the probabilistic decision of a cell to enter in division, rest in quiescence or migrate/die. The modelling analysis indicated that the probability of a cell to proliferate was higher following vaginal than nasal immunization. All together these data show that vaginal immunization, despite the absence of an organized mucosal associated inductive site in the genital tract, is very efficient in priming antigen-specific CD4⁺ T cells and inducing their dissemination from draining lymph nodes towards distal lymphoid organs.     

Structural basis of tubulin tyrosination by tubulin tyrosine ligase

Tubulin tyrosine ligase (TTL) catalyzes the post-translational retyrosination of detyrosinated α-tubulin. Despite the indispensable role of TTL in cell and organism development, its molecular mechanism of action is poorly understood. By solving crystal structures of TTL in complex with tubulin, we here demonstrate that TTL binds to the α and β subunits of tubulin and recognizes the curved conformation of the dimer. Biochemical and cellular assays revealed that specific tubulin dimer recognition controls the activity of the enzyme, and as a consequence, neuronal development. The TTL–tubulin structure further illustrates how the enzyme binds the functionally crucial C-terminal tail sequence of α-tubulin and how this interaction catalyzes the tyrosination reaction. It also reveals how TTL discriminates between α- and β-tubulin, and between different post-translationally modified forms of α-tubulin. Together, our data suggest that TTL has specifically evolved to recognize and modify tubulin, thus highlighting a fundamental role of the evolutionary conserved tubulin tyrosination cycle in regulating the microtubule cytoskeleton.