Chemical rescue of a site-specific mutant of bacterial copper amine oxidase for generation of the topa quinone cofactor

The topa quinone (TPQ) cofactor of copper amine oxidase is produced by posttranslational modification of a specific tyrosine residue through the copper-dependent, self-catalytic process. We have site-specifically mutated three histidine residues (His431, His433, and His592) involved in binding of the copper ion in the recombinant phenylethylamine oxidase from Arthrobacter globiformis. The mutant enzymes, in which each histidine was replaced by alanine, were purified in the Cu/TPQ-free precursor form and analyzed for their Cu-binding and TPQ-generating activities by UV-visible absorption, resonance Raman, and electron paramagnetic resonance spectroscopies. Among the three histidine-to-alanine mutants, only H592A was found to show a weak activity to form TPQ upon aerobic incubation with Cu(2+) ions. Also for H592A, exogenous imidazole rescued binding of copper and markedly promoted the TPQ formation. Accommodation of a free imidazole molecule within the cavity created in the active site of H592A was suggested by X-ray crystallography. Although the TPQ cofactor in H592A mutant was readily reduced with substrate, its catalytic activity was very low even in the presence of imidazole. Combined with the crystal structures of the mutant enzymes, these results demonstrate the importance of the three copper-binding histidine residues for both TPQ biogenesis and catalytic activity, fine-tuning the position of the essential metal.     

In vitro toxicity of palladium(II) and gold(III) porphyrins and their aqueous metal ion counterparts on Trypanosoma brucei brucei growth

The trypanocidal effects of aqueous gold(III) and palladium(II) and their metalloporphyrin derivatives on Trypanosoma brucei brucei growth in culture have been studied using an Alamar Blue indicator assay. All the experiments were conducted in the dark. As previously described for mercury(II), cadmium(II) and lead(II) porphyrins [Chem.-Biol. Interact. 139 (2002) 177], the toxicity of the metalloporphyrin complex of palladium(II) to T. b. brucei parasites was much higher compared to the aqueous free palladium(II) and free base porphyrin. Palladium(II) porphyrin, free palladium(II), and the free base porphyrin were trypanocidal to T. b. brucei at concentrations >1.5 x 10(-6), >6.1 x 10(-6) and >1.9 x 10(-5) M, respectively. While gold(III) porphyrin was effective against the parasites at concentrations >4.8 x 10(-6) M, its aqueous gold(III) was toxic at concentrations as low as 2.0 x 10(-7) M due to the generation of free radicals in the presence of this metal ion which enhanced its toxicity to the T. b. brucei parasites. Although some cell division was observed in some of the cells treated with palladium(II) porphyrin, some dividing cells had no nucleus due to unequal division and delivery of the nuclei into the daughter cells. As a result, the rate of cell division decreased with time and cell death occurred within 24 h. Interestingly, trypanosomes treated with metalloporphyrin complexes displayed different morphological features from those cells treated with free base porphyrin or metal ions. Of all the porphyrins and free metal ions tested, only mercury(II) porphyrin and aqueous gold(III) ion were toxic to the trypanosomes in the 10(-7) M range. The chemotherapeutic potential of these observations is discussed.     

P311 accelerates nerve regeneration of the axotomized facial nerve

In axotomized adult neurons, a process of axonal regrowth and re-establishment of the neuronal function has to be activated. Developmentally regulated factors may be reactivated during neuronal regeneration. Here we identify a gene, previously designated P311, that is up-regulated in the axotomized facial motoneurons. Ectopically expressed P311 localizes in the cytoplasm and the nucleus. Over-expression of P311 induces p21(WAF1/Cip1) expression, leading PC12 cells to differentiate and to have neuron-like morphologies. Adenovirus-mediated P311 gene transfer promotes neurite outgrowth of postnatal dorsal root ganglion neurons and embryonic hippocampal neurons in vitro. This effect is abolished by the activation of Rho kinase. P311 also facilitates nerve regeneration following facial nerve injury in vivo. Our data provide evidence that genes involved in the differentiation process contribute to the regeneration of injured mature neurons, and may provide a practical molecular target.     

African trypanosome interactions with an in vitro model of the human blood-brain barrier

The neurological manifestations of sleeping sickness in man are attributed to the penetration of the blood-brain barrier (BBB) and invasion of the central nervous system by Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. However, how African trypanosomes cross the BBB remains an unresolved issue. We have examined the traversal of African trypanosomes across the human BBB using an in vitro BBB model system constructed of human brain microvascular endothelial cells (BMECs) grown on Costar Transwell inserts. Human-infective T. b. gambiense strain IL 1852 was found to cross human BMECs far more readily than the animal-infective Trypanosoma brucei brucei strains 427 and TREU 927. Tsetse fly-infective procyclic trypomastigotes did not cross the human BMECs either alone or when coincubated with bloodstreamform T. b. gambiense. After overnight incubation, the integrity of the human BMEC monolayer measured by transendothelial electrical resistance was maintained on the inserts relative to the controls when the endothelial cells were incubated with T. b. brucei. However, decreases in electrical resistance were observed when the BMEC-coated inserts were incubated with T. b. gambiense. Light and electron microscopy studies revealed that T. b. gambiense initially bind at or near intercellular junctions before crossing the BBB paracellularly. This is the first demonstration of paracellular traversal of African trypanosomes across the BBB. Further studies are required to determine the mechanism of BBB traversal by these parasites at the cellular and molecular level.     

Analysis of major virulence factors in Porphyromonas gingivalis under various culture temperatures using specific antibodies

Porphyromonas gingivalis is implicated in the occurrence of adult periodontitis. We have previously identified major outer membrane proteins from P. gingivalis, which include representative virulence factors such as gingipains, a 75 kDa major protein, RagA, RagB, and putative porin. Fimbriae, another important virulence factor, exist on the cell surface. In this study, we identified major supernatant proteins. They were fimbrilin, the 75 kDa major protein, gingipains and their adhesin domains. Microscopic examination showed that supernatant proteins formed vesicle-like and fimbrial structures. To learn more about the character of this bacterium, we examined effects of growth temperature on localization and expression of these virulence factors. In general, localization of major virulence factors did not change at the various growth temperatures used. Most of the 75 kDa major protein, RagA, RagB, and putative porin were found in the envelope fraction, not in cell-free culture supernatant. Gingipains were found in both the envelope fraction and supernatant. More than 80% of fimbriae were associated with cells, less than 20% migrated to the supernatant. Most fimbriae existed in the whole cell lysate, although there was a small amount in the envelope fraction. When the growth temperature was increased, expression of fimbriae, gingipains, the 75 kDa major protein, RagA, and RagB decreased. However, temperature had almost no effect on expression of putative porin. The tendency for expression of major virulence factors to decrease at higher temperatures may enable P. gingivalis to survive under hostile conditions.     

Tubulin and CRMP-2 complex is transported via Kinesin-1

The transport of tubulin and microtubules in a growing axon is essential for axonal growth and maintenance. However, the molecular mechanism underlying the linkage of tubulin and microtubules to motor proteins is not yet clear. Collapsin response mediator protein-2 (CRMP-2) is enriched at the distal part of growing axons in primary hippocampal neurons and plays a critical role in axon differentiation through its interaction with tubulin dimer and Numb. In this study, we show that CRMP-2 regulates tubulin transport by linking tubulin and Kinesin-1. The C-terminal region of CRMP-2 directly binds to the tetratricopeptide repeat domain of kinesin light chain 1 (KLC1). Soluble tubulin binds to the middle of CRMP-2 and forms a trimeric complex with CRMP-2/KLC1. Furthermore, the movement of GFP-tubulin in the photobleached area is weakened by knockdown of KLCs or CRMP-2. These results indicate that the CRMP-2/Kinesin-1 complex regulates soluble tubulin transport to the distal part of the growing axon.     

Prostaglandin E2 enhances osteoclastic differentiation of precursor cells through protein kinase A-dependent phosphorylation of TAK1

Prostaglandin E2 (PGE2) synergistically enhances the receptor activator for NF-kappa B ligand (RANKL)-induced osteoclastic differentiation of the precursor cells. Here we investigated the mechanisms of the stimulatory effect of PGE2 on osteoclast differentiation. PGE2 enhanced osteoclastic differentiation of RAW264.7 cells in the presence of RANKL through EP2 and EP4 prostanoid receptors. RANKL-induced degradation of I kappa B alpha and phosphorylation of p38 MAPK and c-Jun N-terminal kinase in RAW264.7 cells were up-regulated by PGE2 in a cAMP-dependent protein kinase A (PKA)-dependent manner, suggesting that EP2 and EP4 signals cross-talk with RANK signals. Transforming growth factor beta-activated kinase 1 (TAK1), an important MAPK kinase kinase in several cytokine signals, possesses a PKA recognition site at amino acids 409-412. PKA directly phosphorylated TAK1 in RAW264.7 cells transfected with wild-type TAK1 but not with the Ser412 --> Ala mutant TAK1. Ser412 --> Ala TAK1 served as a dominant-negative mutant in PKA-enhanced degradation of I kappa B alpha, phosphorylation of p38 MAPK, and PGE2-enhanced osteoclastic differentiation in RAW264.7 cells. Furthermore, forskolin enhanced tumor necrosis factor alpha-induced I kappa B alpha degradation, p38 MAPK phosphorylation, and osteoclastic differentiation in RAW264.7 cells. Ser412 --> Ala TAK1 abolished the stimulatory effects of forskolin on those cellular events induced by tumor necrosis factor alpha. Ser412 --> Ala TAK1 also inhibited the forskolin-induced up-regulation of interleukin 6 production in RAW264.7 cells treated with lipopolysaccharide. These results suggest that the phosphorylation of the Ser412 residue in TAK1 by PKA is essential for cAMP/PKA-induced up-regulation of osteoclastic differentiation and cytokine production in the precursor cells.     

Protein kinase Ctheta activity is involved in the 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced signal transduction pathway leading to apoptosis in L-MAT, a human lymphoblastic T-cell line

The aromatic hydrocarbon receptor (AhR)-dependent pathway involved in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced immunotoxicity has been studied extensively, but the AhR-independent molecular mechanism has not. In previous studies we found that the AhR is not expressed in L-MAT, a human lymphoblastic T-cell line. In this report, we provide the following evidence that the protein kinase C (PKC)theta activity is functionally involved in the AhR-independent signal transduction mechanism that participates in the TCDD-induced L-MAT cell apoptosis. First, only rottlerin, a novel PKC (nPKC)-selective inhibitor, blocked the apoptosis completely, in a dose-dependent manner. Second, PKCtheta was the major nPKC isoform (compared to PKCdelta) expressed in the L-MAT cell line. Third, a cell-permeable myristoylated PKCtheta pseudosubstrate peptide inhibitor also blocked the apoptosis completely, in a dose-dependent manner. Fourth, both rottlerin and myristoylated PKCtheta pseudosubstrate peptide inhibitor completely inhibited PKCtheta kinase activity in vitro at doses that effectively blocked TCDD-induced L-MAT cell apoptosis. TCDD treatment induced a time-dependent activation of nPKC kinase activity in L-MAT cells, and moreover, TCDD induced a translocation of PKCtheta from the cytosolic fraction to the particulate fraction in L-MAT cells. Finally, transient over-expression of a dominant negative PKCtheta (a kinase-dead mutant, K/R 409) in L-MAT cells conferred significant protection against TCDD-induced apoptosis.