Stoichiometric relations in an ant-treehopper mutualism

Carbon : nitrogen : phosphorus (C : N : P) stoichiometry can underlie physiological and life history characteristics that shape ecological interactions. Despite its potential importance, there is much to learn about the causes and consequences of stoichiometric variation in terrestrial consumers. Here we show that treehoppers (Publilia modesta) tended by ants (Formica obscuripes) contained lower N concentrations than treehoppers on plants from which ants were excluded. Ant presence also affected nutrient concentrations in host plants: on plants with ants, leaves contained uniformly low concentrations of N; on plants without ants, N concentrations were low only in the few leaves fed upon by treehoppers at the time of collection. We suggest treehopper feeding reduces leaf nutrient levels and ants positively affect treehopper abundance, producing a top–down effect on plant quality. Determining the causes of these stoichiometric changes should help elucidate factors guiding the dynamics of conditional mutualisms between ants and homopterans.     

The oxygenase component of phenol hydroxylase from Acinetobacter radioresistens S13.

Phenol hydroxylase (PH) from Acinetobacter radioresistens S13 represents an example of multicomponent aromatic ring monooxygenase made up of three moieties: a reductase (PHR), an oxygenase (PHO) and a regulative component (PHI). The function of the oxygenase component (PHO), here characterized for the first time, is to bind molecular oxygen and catalyse the mono-hydroxylation of substrates (phenol, and with less efficiency, chloro- and methyl-phenol and naphthol). PHO was purified from extracts of A. radioresistens S13 cells and shown to be a dimer of 206 kDa. Each monomer is composed by three subunits: alpha (54 kDa), beta (38 kDa) and gamma (11 kDa). The gene encoding PHO alpha (named mopN) was cloned and sequenced and the corresponding amino acid sequence matched with that of functionally related oxygenases. By structural alignment with the catalytic subunits of methane monooxygenase (MMO) and alkene monooxygenase, we propose that PHO alpha contains the enzyme active site, harbouring a dinuclear iron centre Fe-O-Fe, as also suggested by spectral analysis. Conserved hydrophobic amino acids known to define the substrate recognition pocket, are also present in the alpha-subunit. The prevalence of alpha-helices (99.6%) as studied by CD confirmed the hypothized structural homologies between PHO and MMO. Three parameters (optimum ionic strength, temperature and pH) that affect kinetics of the overall phenol hydroxylase reaction were further analyzed with a fixed optimal PHR/PHI/PHO ratio of 2/1/1. The highest level of activity was evaluated between 0.075 and 0.1 m of ionic strength, the temperature dependence showed a maximum of activity at 24 degrees C and finally the pH for optimal activity was determined to be 7.5.     

Comparative importance in vivo of conserved glutamate residues in the EX7E motif retaining glycosyltransferase Gpi3p, the UDP-GlcNAc-binding subunit of the first enzyme in glycosylphosphatidylinositol assembly.

Saccharomyces cerevisiae Gpi3p is the UDP-GlcNAc-binding and presumed catalytic subunit of the enzyme that forms GlcNAc-phosphatidylinositol in glycosylphosphatidylinositol biosynthesis. It is an essential protein with an EX7E motif that is conserved in four families of retaining glycosyltransferases. All Gpi3ps contain a cysteine residue four residues C-terminal to EX7E. To test their importance for Gpi3p function in vivo, Glu289 and 297 in the EX7E motif of S. cerevisiae Gpi3p, as well as Cys301, were altered by site-specific mutagenesis, and the mutant proteins tested for their ability to complement nonviable GPI3-deleted haploids. Gpi3p-C301A supported growth but membranes from C301A-expressing cells had low in vitro N-acetylglucosaminylphosphatidylinositol (GlcNAc-PI) synthetic activity. Haploids harboring Gpi3p-E289A proved viable, although slow growing but Gpi3-E297A did not support growth. The E289D and E297D mutants both supported growth at 25 degrees C, but, whereas the E289D strain grew at 37 degrees C, the E297D mutant did not. Membranes from E289D mutants had severely reduced in vitro GlcNAc-PI synthetic activity and E297D membranes had none. The mutation of the first Glu in the EX7E motif of Schizosaccharomyces pombe Gpi3p (Glu277) to Asp complemented the lethal null mutation in gpi3+ and supported growth at 37 degrees C, but the E285D mutant was nonviable. Our results suggest that the second Glu residue of the EX7E motif in Gpi3p is of greater importance than the first for function in vivo. Further, our findings do not support previous suggestions that the first Glu of an EX7E protein is the nucleophile and that Cys301 has an important role in UDP-GlcNAc binding by Gpi3ps.     

1α,25-dihydroxy-24-oxo-16-ene vitamin D3, a metabolite of a synthetic vitamin D3 analog, 1α,25-dihydroxy-16-ene vitamin D3, is equipotent to its parent in modulating growth and differentiation of human leukemic cells

1α,25(OH)₂-16-ene-D₃, a synthetic analog of the steroid hormone, 1α,25(OH)₂D₃, has great potential to become a drug in the treatment of leukemia and other proliferative disorders, because of its minimal in vivo calcemic activity associated with a potent inhibitory effect on cell growth. However, at present, the mechanisms through which 1α,25(OH)₂-16-ene-D₃ expresses its biological activities are still not completely understood. Our previous in vitro study in a perfused rat kidney indicated for the first time that 1α,25(OH)₂-16-ene-D₃ and 1α,25(OH)₂D₃ are metabolized differently. 1α,25(OH)₂-24-oxo-16-ene-D₃, an intermediary metabolite of 1α,25(OH)₂-16-ene-D₃ formed through the C-24 oxidation pathway, accumulated significantly in the perfusate when compared to 1α,25(OH)₂-24-oxo-D₃, the corresponding intermediary metabolite of 1α,25(OH)₂D₃. In a subsequent in vivo study, we also reported that 1α,25(OH)₂-24-oxo-16-ene-D₃ exerted immunosuppressive activity equal to its parent, without causing significant hypercalcemia. In order to establish further the critical role of 1α,25(OH)₂-24-oxo-16-ene-D₃, in generating some of the key biological activities ascribed to its parent, we performed the present in vitro study using a human myeloid leukemic cell line (RWLeu-4) as a model. Comparative target tissue metabolism studies indicated that 1α,25(OH)₂-16-ene-D₃ and 1α,25(OH)₂D₃ are metabolized differently in RWLeu-4 cells, and the differences were similar to the ones we previously observed in the rat kidney. The significant finding was the accumulation of 1α,25(OH)₂-24-oxo-16-ene-D₃ in RWLeu-4 cells because of its resistance to further metabolism. Biological activity studies indicated that both 1α,25(OH)₂-16-ene-D₃ and its 24-oxo metabolite produced growth inhibition and promoted differentiation of RWLeu-4 cells to the same extent, and these activities were several fold higher than those exerted by 1α,25(OH)₂D₃. In addition, the genomic action of each vitamin D compound was assessed in a rat osteosarcoma cell line (ROS 17/2.8) by measuring its ability to transactivate a gene construct containing the vitamin D response element of the osteocalcin gene linked to the growth hormone reporter gene. In these studies, both 1α,25(OH)₂-16-ene-D₃ and its 24-oxo metabolite exerted similar but potent transactivation activity which was several fold greater than that exerted by 1α,25(OH)₂D₃ itself. In summary, our results indicate that the production and slow clearance of the bioactive intermediary metabolite, 1α,25(OH)₂-24-oxo-16-ene-D₃, in RWLeu-4 cells contributes significantly to the final expression of the enhanced biological activities ascribed to its parent analog, 1α,25(OH)₂-16-ene-D₃.     

Efficiency of reinitiation of translation on human immunodeficiency virus type 1 mRNAs is determined by the length of the upstream open reading frame and by intercistronic distance.

In this study, we examined the mechanism of translation of the human immunodeficiency virus type 1 tat mRNA in eucaryotic cells. This mRNA contains the tat open reading frame (ORF), followed by rev and nef ORFs, but only the first ORF, encoding tat, is efficiently translated. Introduction of premature stop codons in the tat ORF resulted in efficient translation of the downstream rev ORF. We show that the degree of inhibition of translation of rev is proportional to the length of the upstream tat ORF. An upstream ORF spanning 84 nucleotides was predicted to inhibit 50% of the ribosomes from initiating translation at downstream AUGs. Interestingly, the distance between the upstream ORF and the start codon of the second ORF also played a role in efficiency of downstream translation initiation. It remains to be investigated if these conclusions relate to translation of mRNAs other than human immunodeficiency virus type 1 mRNAs. The strong inhibition of rev translation exerted by the presence of the tat ORF may reflect the different roles of Tat and Rev in the viral life cycle. Tat acts early to induce high production of all viral mRNAs. Rev induces a switch from the early to the late phase of the viral life cycle, resulting in production of viral structural proteins and virions. Premature Rev production may result in entrance into the late phase in the presence of suboptimal levels of viral mRNAs coding for structural proteins, resulting in inefficient virus production.     

Accumulation of wild-type p53 protein upon gamma-irradiation induces a G2 arrest-dependent immunoglobulin kappa light chain gene expression.

The exposure of cells to DNA-damaging agents leads to the accumulation of wild-type p53 protein. Furthermore, overexpression of the wild-type p53, mediated by transfection of p53-coding cDNA, induced cells to undergo apoptosis or cell differentiation. In this study we found that the gamma-irradiation that caused the accumulation of wild-type p53 in 70Z/3 pre-B cells induced, in addition to apoptosis, cell differentiation. This was manifested by the expression of the kappa light chain immunoglobulin gene that coincided with the accumulation of cells at the G2 phase. Overexpression of mutant p53 in 70Z/3 cells interferes with both differentiation and accumulation of cells at the G2 phase, as well as with apoptosis, which were induced by gamma-irradiation. Furthermore, the increment in the wild-type p53 protein level following gamma-irradiation was disrupted in the mutant p53 overproducer-derived cell lines. This suggests that mutant p53 may exert a dominant negative effect in all of these activities. Data presented here show that while p53-induced apoptosis is associated with the G1 checkpoint, p53-mediated differentiation, which may be an additional pathway to escape the fixation of genetic errors, may be associated with the G2 growth arrest phase.     

Charge transfer photochemistry of quadruply bonded ditungsten halophosphine complexes

The quadruple metal-metal bonded complexes, W₂Cl₄(PR₃)₄ (PR₃ = PMe₃, PMe₂Ph, PBu₃), photoreact in dichloromethane with near-UV excitation (λ>375 nm) to yield a mixed valence W₂(II,III) photoproduct. Electronic absorption and EPR spectra of photolyzed solutions are identical to those obtained from the thermal oxidation of W₂Cl₄(PR₃)₄ by PhICI₂, which is known to yield W₂Cl₅(PR₃)₃. Subsequent reaction of the photolyzed solution yields the oxidized, confacial biotahedral W₂(III,III) halophosphine. Analysis of the organic photoproduct reveals that the halocarbon solvent is reduced by one electron to yield the chloromethyl radical. When the radical is produced in low yields, hydrogen abstraction from solvent appears to be sufficiently efficient to compete with dimerization and only chloromethane is observed; however, at higher concentrations, the chloromethyl radicals couple to produce dichloroethane. Photoreaction is observed only with near-UV excitation of the LMCT absorption manifold of W₂Cl₄(PR₃)₄. At lower energy wavelengths, transient absorption spectroscopy shows the production of the ¹δδ^* excited state, which decays to ground state over times commensurate with the decay of ¹δδ^* luminescence. In hydrocarbon solutions, no transient intermediate or photochemistry is observed, indicating that the LMCT excited state, although capable of reducing a C---X bond, cannot activate the stronger C---H bonds of hydrocarbons. The photochemistry and transient absorption spectroscopy results of the W₂Cl₄(PR₃)₄ complexes are compared to our previous studies of the homologs.