Phylogeny of the Celastraceae inferred from phytochrome B gene sequence and morphology

Phylogenetic relationships within Celastraceae were inferred using a simultaneous analysis of 61 morphological characters and 1123 base pairs of phytochrome B exon 1 from the nuclear genome. No gaps were inferred, and the gene tree topology suggests that the primers were specific to a single locus that did not duplicate among the lineages sampled. This region of phytochrome B was most useful for examining relationships among closely related genera. Fifty-one species from 38 genera of Celastraceae were sampled. The Celastraceae sensu lato (including Hippocrateaceae) were resolved as a monophyletic group. Loesener's subfamilies and tribes of Celastraceae were not supported. The Hippocrateaceae were resolved as a monophyletic group nested within a paraphyletic Celastraceae sensu stricto. Goupia was resolved as more closely related to Euphorbiaceae, Corynocarpaceae, and Linaceae than to Celastraceae. Plagiopteron (Flacourtiaceae) was resolved as the sister group of Hippocrateoideae. Brexia (Brexiaceae) was resolved as closely related to Elaeodendron and Pleurostylia. Canotia was resolved as the sister group of Acanthothamnus within Celastraceae. Perrottetia and Mortonia were resolved as the sister group of the rest of the Celastraceae. Siphonodon was resolved as a derived member of Celastraceae. Maytenus was resolved as three disparate groups, suggesting that this large genus needs to be recircumscribed.     

Using an RNA secondary structure partition function to determine confidence in base pairs predicted by free energy minimization

A partition function calculation for RNA secondary structure is presented that uses a current set of nearest neighbor parameters for conformational free energy at 37 degrees C, including coaxial stacking. For a diverse database of RNA sequences, base pairs in the predicted minimum free energy structure that are predicted by the partition function to have high base pairing probability have a significantly higher positive predictive value for known base pairs. For example, the average positive predictive value, 65.8%, is increased to 91.0% when only base pairs with probability of 0.99 or above are considered. The quality of base pair predictions can also be increased by the addition of experimentally determined constraints, including enzymatic cleavage, flavin mono-nucleotide cleavage, and chemical modification. Predicted secondary structures can be color annotated to demonstrate pairs with high probability that are therefore well determined as compared to base pairs with lower probability of pairing.     

Chemical and kinetic reaction mechanisms of quinohemoprotein amine dehydrogenase from Paracoccus denitrificans

Quinohemoprotein amine dehydrogenase (QHNDH) possesses a cysteine tryptophylquinone (CTQ) prosthetic group that catalyzes the oxidative deamination of primary amines. In addition to CTQ, two heme c cofactors are present in QHNDH that mediate the transfer of the substrate-derived electrons from CTQ to an external electron acceptor. Steady-state kinetic assays yielded relatively small k(cat) values (<6 s(-1)), and the rate-limiting step appears to be the interprotein electron transfer from heme in QHNDH to the external electron acceptor. Transient kinetic studies of the CTQ-dependent reduction of heme in QHNDH by amine substrates yielded different rate constants for different substrates (72, 190, and 162 s(-1) for methylamine, butylamine, and benzylamine, respectively). Deuterium kinetic isotope effect (KIE) values of 5.3, 3.9, and 8.5 were observed, respectively, for the reactions of methylamine, butylamine, and benzylamine. These results suggest that the abstraction of a proton from the alpha-methylene group of the substrate, which occurs concomitant with CTQ reduction, is the rate-limiting step in the CTQ-dependent reduction of hemes in QHNDH by these amine substrates. In contrast, the reaction of 2-phenylethylamine with QHNDH does not exhibit a significant KIE ((H)k(3)/(D)k(3) = 1.05) and exhibits a much smaller rate constant of 16 s(-1). This suggests that for 2-phenylethylamine, the rate-limiting step in the single-turnover reaction is either hydrolysis of the imine reaction intermediate from CTQ or product release prior to intraprotein electron transfer. Analysis of the products of the reactions of QHNDH with chiral deuterated 2-phenylethylamines demonstrated that the enzyme abstracts the pro-S proton of the substrate in a highly stereospecific manner. Inspection of the crystal structure of phenylhydrazine-inhibited QHNDH suggests that Asp33(gamma) is the residue that performs the proton abstraction. On the basis of these results, kinetic and chemical reaction mechanisms for QHNDH are proposed and discussed in the context of the crystal structure of the enzyme.     

Phospholipase C-gamma contains introns shared by src homology 2 domains in many unrelated proteins

Many proteins with novel functions were created by exon shuffling around the time of the metazoan radiation. Phospholipase C-gamma (PLC-gamma) is typical of proteins that appeared at this time, containing several different modules that probably originated elsewhere. To gain insight into both PLC-gamma evolution and structure-function relationships within the Drosophila PLC-gamma encoded by small wing (sl), we cloned and sequenced the PLC-gamma homologs from Drosophila pseudoobscura and D. virilis and compared their gene structure and predicted amino acid sequences with PLC-gamma homologs in other animals. PLC-gamma has been well conserved throughout, although structural differences suggest that the role of tyrosine phosphorylation in enzyme activation differs between vertebrates and invertebrates. Comparison of intron positions demonstrates that extensive intron loss has occurred during invertebrate evolution and also reveals the presence of conserved introns in both the N- and C-terminal PLC-gamma SH2 domains that are present in SH2 domains in many other genes. These and other conserved SH2 introns suggest that the SH2 domains in PLC-gamma are derived from an ancestral domain that was shuffled not only into PLC-gamma, but also into many other unrelated genes during animal evolution.     

Deoxyribonucleotide pool imbalance stimulates deletions in HeLa cell mitochondrial DNA

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder associated with multiple mutations in mitochondrial DNA, both deletions and point mutations, and mutations in the nuclear gene for thymidine phosphorylase. Spinazzola et al. (Spinazzola, A., Marti, R., Nishino, I., Andreu, A., Naini, A., Tadesse, S., Pela, I., Zammarchi, E., Donati, M., Oliver, J., and Hirano, M. (2001) J. Biol. Chem. 277, 4128-4133) showed that MNGIE patients have elevated circulating thymidine levels and they hypothesized that this generates imbalanced mitochondrial deoxyribonucleoside triphosphate (dNTP) pools, which in turn are responsible for mitochondrial (mt) DNA mutagenesis. We tested this hypothesis by culturing HeLa cells in medium supplemented with 50 microM thymidine. After 8-month growth, mtDNA in the thymidine-treated culture, but not the control, showed multiple deletions, as detected both by Southern blotting and by long extension polymerase chain reaction. After 4-h growth in thymidine-supplemented medium, we found the mitochondrial dTTP and dGTP pools to expand significantly, the dCTP pool to drop significantly, and the dATP pool to drop slightly. In whole-cell extracts, dTTP and dGTP pools also expanded, but somewhat less than in mitochondria. The dCTP pool shrank by about 50%, and the dATP pool was essentially unchanged. These results are discussed in terms of the recent report by Nishigaki et al. (Nishigaki, Y., Marti, R., Copeland, W. C., and Hirano, M. (2003) J. Clin. Invest. 111, 1913-1921) that most mitochondrial point mutations in MNGIE patients involve T --> C transitions in sequences containing two As to the 5' side of a T residue. Our finding of dTTP and dGTP elevations and dATP depletion in mitochondrial dNTP pools are consistent with a mutagenic mechanism involving T-G mispairing followed by a next-nucleotide effect involving T insertion opposite A.     

Alterations in the GnRH-LH system in relation to gonadal stage and Aroclor 1254 exposure in Atlantic croaker

Exposure of Atlantic croaker (Micropogonias undulatus) to the polychlorinated biphenyl mixture (Aroclor 1254, PCB; 1 mg/kg body wt/day for 30 days) during the early-recrudescence phase of the gonadal cycle results in the impairment of LH secretion and gonadal growth. In order to determine whether impairment was due to disruption of the stimulatory GnRH neuroendocrine pathway, we compared various parameters of the GnRH-LH system in early recrudescing vs. spermiating (mature) fish. Seabream GnRH (GnRH) content in the preoptic anterior hypothalamic area (POAH) and pituitary, pituitary GnRH receptor concentrations, and basal and GnRH analog (GnRHa)-induced LH secretion were significantly higher in gonadally mature croaker compared to early-recrudescing fish. In a subsequent experiment, the effects of PCB on the same neuroendocrine indices were investigated during the gonadal recrudescence phase of croaker. PCB exposure during the period of testicular maturation prevented the natural increase in GnRH content in the POAH but not in the pituitary. This finding suggests that PCB may impair GnRH synthesis in the POAH. The number of pituitary GnRH receptors also remained significantly lower in the PCB-exposed group, which was likely due to an impairment of GnRH release. The GnRH content in the POAH, number of pituitary GnRH receptors, and LH secretion in the PCB-exposed group were comparable to those in early-recrudescing fish, suggesting an impairment of normal maturation of the GnRH-LH system during the gonadal recrudescence phase. This impairment may be due to a direct action of PCB on GnRH neurons and/or indirectly via interference with other neurotransmitter pathways that modulate GnRH function.