Metabolism of Melamine by Klebsiella terragena

Experiments were conducted to determine the pathway of melamine metabolism by Klebsiella terragena (strain DRS-1) and the effect of added NH(inf4)(sup+) on the rates and extent of melamine metabolism. In the absence of added NH(inf4)(sup+), 1 mM melamine was metabolized concomitantly with growth. Ammeline, ammelide, cyanuric acid, and NH(inf4)(sup+) accumulated transiently in the culture medium to maximal concentrations of 0.012 mM, 0.39 mM, trace levels, and 0.61 mM, respectively. In separate incubations, in which cells were grown on either ammeline or ammelide (in the absence of NH(inf4)(sup+)), ammeline was metabolized without a lag while ammelide metabolism was observed only after 3 h. In the presence of 6 mM added NH(inf4)(sup+) (enriched with 5% (sup15)N), ammeline, ammelide, and cyanuric acid accumulated transiently to maximal concentrations of 0.002 mM, 0.47 mM, and trace levels, respectively, indicating that the added NH(inf4)(sup+) had little effect on the relative rates of triazine metabolism. These data suggest that the primary mode of melamine metabolism by K. terragena is hydrolytic, resulting in successive deaminations of the triazine ring. Use of (sup15)N-enriched NH(inf4)(sup+) allowed estimates of rates of triazine-N mineralization and assimilation of NH(inf4)(sup+)-N versus triazine-N into biomass. A decrease in the percent (sup15)N in the external NH(inf4)(sup+) pool, in conjunction with the accumulation of ammelide and/or triazine-derived NH(inf4)(sup+) in the culture medium, suggests that the initial reactions in the melamine metabolic pathway may occur outside the cytoplasmic membrane.     

Chromatin assembly in isolated mammalian nuclei.

Cellular DNA replication was stimulated in confluent monolayers of CV-1 monkey kidney cells following infection with SV40. Nuclei were isolated from CV-1 cells labeled with [3H]thymidine and then incubated in the presence of [alpha-32P]deoxyribonucleoside triphosphates under conditions that support DNA replication. To determine whether or not the cellular DNA synthesized in vitro was assembled into nucleosomes the DNA was digested in situ with either micrococcal nuclease or pancreatic DNase I, and the products were examined by electrophoretic and sedimentation analysis. The distribution of DNA fragment lengths on agarose gels following micrococcal nuclease digestion was more heterogeneous for newly replicated than for the bulk of the DNA. Nonetheless, the state of cellular DNA synthesized in vitro (32P-labeled) was found to be identical with that of the DNA in the bulk of the chromatin (3H-labeled) by the following criteria: (i) The extent of protection against digestion by micrococcal nuclease of DNase I. (ii) The size of the nucleosomes (180 base pairs) and core particles (145 base pairs). (iii) The number and sizes of DNA fragments produced by micrococcal nuclease in a limit digest. (iv) The sedimentation behavior on neutral sucrose gradients of nucleoprotein particles released by micrococcal nuclease. (v) The number and sizes of DNA fragments produced by DNase I digestion. These results demonstrate that cellular DNA replicated in isolated nuclei is organized into typical nucleosomes. Consequently, subcellular systems can be used to study the relationship between DNA replication and the assembly of chromatin under physiological conditions.     

Slow Replication Fork Velocity of Homologous Recombination-Defective Cells Results from Endogenous Oxidative Stress

Replications forks are routinely hindered by different endogenous stresses. Because homologous recombination plays a pivotal role in the reactivation of arrested replication forks, defects in homologous recombination reveal the initial endogenous stress(es). Homologous recombination-defective cells consistently exhibit a spontaneously reduced replication speed, leading to mitotic extra centrosomes. Here, we identify oxidative stress as a major endogenous source of replication speed deceleration in homologous recombination-defective cells. The treatment of homologous recombination-defective cells with the antioxidant N-acetyl-cysteine or the maintenance of the cells at low O₂ levels (3%) rescues both the replication fork speed, as monitored by single-molecule analysis (molecular combing), and the associated mitotic extra centrosome frequency. Reciprocally, the exposure of wild-type cells to H₂O₂ reduces the replication fork speed and generates mitotic extra centrosomes. Supplying deoxynucleotide precursors to H₂O₂-exposed cells rescued the replication speed. Remarkably, treatment with N-acetyl-cysteine strongly expanded the nucleotide pool, accounting for the replication speed rescue. Remarkably, homologous recombination-defective cells exhibit a high level of endogenous reactive oxygen species. Consistently, homologous recombination-defective cells accumulate spontaneous γH2AX or XRCC1 foci that are abolished by treatment with N-acetyl-cysteine or maintenance at 3% O₂. Finally, oxidative stress stimulated homologous recombination, which is suppressed by supplying deoxynucleotide precursors. Therefore, the cellular redox status strongly impacts genome duplication and transmission. Oxidative stress should generate replication stress through different mechanisms, including DNA damage and nucleotide pool imbalance. These data highlight the intricacy of endogenous replication and oxidative stresses, which are both evoked during tumorigenesis and senescence initiation, and emphasize the importance of homologous recombination as a barrier against spontaneous genetic instability triggered by the endogenous oxidative/replication stress axis.     

Heavy-metal-contaminated industrial soil: Uptake assessment in native plant species from Brazilian Cerrado

Plants of the Cerrado have shown some potential for restoration and/or phytoremediation projects due to their ability to grow in and tolerate acidic soils rich in metals. The aim of this study is to evaluate the tolerance and accumulation of metals (Cd, Cu, Pb, and Zn) in five native tree species of the Brazilian Cerrado (Copaifera langsdorffii, Eugenia dysenterica, Inga laurina, Cedrela fissilis, Handroanthus impetiginosus) subjected to three experiments with contaminated soils obtained from a zinc processing industry (S₁, S₂, S₃) and control soil (S₀). The experimental design was completely randomized (factorial 5 × 4 × 3) and conducted in a greenhouse environment during a 90-day experimentation time. The plant species behavior was assessed by visual symptoms of toxicity, tolerance index (TI), translocation factor (TF), and bioaccumulation factor (BF). C. fissilis has performed as a Zn accumulator by the higher BFs obtained in the experiments, equal to 3.72, 0.88, and 0.41 for S₁, S₂, and S₃ respectively. This species had some ability of uptake control as a defense mechanism in high stress conditions with the best behavior for phytoremediation and high tolerance to contamination. With economical and technical benefits, this study may support a preliminary analysis necessary for using native tree species in environmental projects.     

N-(cyclohexanecarboxyl)-O-phospho-l-serine, a minimal substrate for the dual-specificity protein phosphatase IphP

Three dual-specific phosphatases [DSPs], IphP, VHR, and Cdc14, and three protein-tyrosine phosphatases [PTPs], PTP-1B, PTP-H1, and Tc-PTPa, were challenged with a set of low molecular weight phosphoesters to probe the factors underlying the distinct substrate specificities displayed by these two mechanistically homologous families of protein phosphatases. It was observed that beta-naphthyl phosphate represented an excellent general substrate for both PTPs and DSPs. While DSPs tended to hydrolyze alpha-naphthyl phosphate at rates comparable to that of the beta-isomer, the PTPs PTP-1B and Tc-PTPa did not. PTP-H1, however, displayed high alpha-naphthyl phosphatase activity. Intriguingly, PTP-H1 also displayed much higher protein-serine phosphatase activity in vitro, 0.2-0.3% that toward equivalent tyrosine phosphorylated proteins, than did PTP-1B or Tc-PTPa. The latter two PTPs discriminated between the serine- and tyrosine-phosphorylated forms of two test proteins by factors of >/=10(4)-10(6). While free phosphoserine represented an extremely poor substrate for all of the DSPs examined, the addition of a hydrophobic "handle" to form N-(cyclohexanecarboxyl)-O-phospho-l-serine produced a compound that was hydrolyzed by IphP with high efficiency, i.e., at a rate comparable to that of free phosphotyrosine or p-nitrophenyl phosphate. VHR also hydrolyzed N-(cyclohexanecarboxyl)-O-phospho-l-serine (1 mM) at a rate approximately one-tenth that of beta-naphthyl phosphate. None of the PTPs tested exhibited significant activity against this compound. However, N-(cyclohexanecarboxyl)-O-phospho-l-serine did not prove to be a universal substrate for DSPs as Cdc14 displayed little propensity to hydrolyze it.     

Applicability of urea in the thermodynamic analysis of secondary and tertiary RNA folding

The equilibrium folding of a series of self-complementary RNA duplexes and the unmodified yeast tRNA(Phe) is studied as a function of urea and Mg(2+) concentration with optical spectroscopies and chemical modification under isothermal conditions. Via application of standard methodologies from protein folding, the folding free energy and its dependence on urea concentration, the m value, are determined. The free energies of the RNA duplexes obtained from the urea titrations are in good agreement with those calculated from thermal melting studies [Freier, S. I., et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 9373]. The m value correlates with the length of the RNA duplex and is not sensitive to ionic conditions and temperature. The folding of the unmodified yeast tRNA(Phe) can be described by two Mg(2+)-dependent transitions, the second of which corresponds to the formation of the native tertiary structure as confirmed by hydroxyl radical protection and partial nuclease digestion. Both transitions are sensitive to urea and have m values of 0.94 and 1.70 kcal mol(-)(1) M(-)(1), respectively. Although the precise chemical basis of urea denaturation of RNA is uncertain, the m values for the duplexes and tRNA(Phe) are proportional to the amount of the surface area buried in the folding transition. This proportionality, 0.099 cal mol(-)(1) M(-)(1) A(-)(2), is very similar to that observed for proteins, 0.11 cal mol(-)(1) M(-)(1) A(-)(2) [Myers, J., Pace, N., and Scholtz, M. (1995) Protein Sci. 4, 2138]. These results indicate that urea titration can be used to measure both the free energy and the magnitude of an RNA folding transition.     

Different cross-resistance patterns in the diamondback moth (Lepidoptera: Plutellidae) resistant to Bacillus thuringiensis toxin Cry1C.

Two strains of the diamondback moth, Plutella xylostella (L.), were selected using Cry1C protoxin and transgenic broccoli plants expressing a Cry1C toxin of Bacillus thuringiensis (Bt). Both strains were resistant to Cry1C but had different cross-resistance patterns. We used 12 Bt protoxins for cross-resistance tests, including Cry1Aa, Cry1Ab, Cry1Ac, Cry1Bb, Cry1C, Cry1D, Cry1E, Cry1F, Cry1J, Cry2Ab, Cry9Aa, and Cry9C. Compared with the unselected sister strain (BCS), the resistance ratio (BR) of one strain (BCS-Cry1C-1) to the Cry1C protoxin was 1,090-fold with high level of cross-resistance to Cry1Aa, Cry1Ab, Cry1Ac, Cry1F, and Cry1J (RR > 390-fold). The cross-resistance to Cry1A, Cry1F, and Cry1J in this strain was probably related to the Cry1A resistance gene(s) that came from the initial field population and was caused by intensive sprayings of Bt products containing Cry1A protoxins. The neonates of this strain can survive on transgenic broccoli plants expressing either Cry1Ac or Cry1C toxins. The other strain (BCS-Cry1C-2) was highly resistant to Cry1C but not cross-resistant to other Bt protoxins. The neonates of this strain can survive on transgenic broccoli expressing Cry1C toxin but not Cry1Ac toxin. The gene(s) conferring resistance to Cry1C segregates independently from Cry1Ac resistance in these strains. The toxicity of Cry1E and Cry2Ab protoxins was low to all of the three strains. The overall progress of all work has resulted in a unique model system to test the stacked genes strategy for resistance management of Bt transgenic crops.