Estimation of the yield coefficient of Pseudomonas sp. strain DP-4 with a low substrate (2,4-dichlorophenol [DCP]) concentration in a mineral medium from which uncharacterized organic compounds were eliminated by a non-DCP-degrading organism.
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The yield coefficient (YC) of Pseudomonas sp. strain DP-4, a 2, 4-dichlorophenol (DCP)-degrading organism, was estimated from the number of CFU produced at the expense of 1 unit amount of DCP at low concentrations. At a low concentration of DCP, the YC can be overestimated in pure culture, because DP-4 assimilated not only DCP but also uncharacterized organic compounds contaminating a mineral salt medium. The concentration of these uncharacterized organic compounds was nutritionally equivalent to 0.7 microg of DCP-C ml(-1). A mixed culture with non-DCP-degrading organisms resulted in elimination of ca. 99.9% of the uncharacterized organic compounds, and then DP-4 assimilated only DCP as a substrate. In a mixed culture, DP-4 degraded an initial concentration of 0.1 to 10 microg of C ml of DCP(-1) and the number of CFU of DP-4 increased. In the mixed culture, DCP at an initial concentration of 0.07 microg of C ml(-1) was degraded. However, the number of CFU of DP-4 did not increase. DCP at an extremely low initial concentration of 0.01 microg of C ml(-1) was not degraded in mixed culture even by a high density, 10(5) CFU ml(-1), of DP-4. When glucose was added to this mixed culture to a final concentration of 1 microg of C ml(-1), the initial concentration of 0.01 microg of C ml of DCP(-1) was degraded. These results suggested that DP-4 required cosubstrates to degrade DCP at an extremely low initial concentration of 0.01 microg of C ml(-1). The YCs of DP-4 at the expense of DCP alone decreased discontinuously with the decrease of the initial concentration of DCP, i.e., 1.5, 0.19, or 0 CFU per pg of DCP-C when 0.7 to 10, 0.1 to 0.5, or 0.07 microg of C ml of DCP(-1) was degraded, respectively. In this study, we developed a new method to eliminate uncharacterized organic compounds, and we estimated the YC of DP-4 at the expense of DCP as a sole source of carbon. (+info)
Density functional theory for the nonspecific binding of salt to polyelectrolytes: thermodynamic properties.
(34/1497)
The thermodynamics of the nonspecific binding of salt to a polyelectrolyte molecule is studied using a density functional approach. The polyelectrolyte molecule is modeled as an infinite, inflexible, and impenetrable charged cylinder and the counterions and co-ions are modeled as charged hard spheres of equal diameter. The density functional theory is based on a hybrid approach where the hard-sphere contribution to the one-particle correlation function is evaluated nonperturbatively and the ionic contribution to the one-particle correlation function is evaluated perturbatively. The advantage of the approach is that analytical expressions are available for all the correlation functions. The calculated single ion preferential interaction coefficients, excess free energy, and activity coefficients show a nonmonotonic variation as a function of polyion charge in the presence of divalent ions. These properties display considerable departure from the predictions of the nonlinear Poisson-Boltzmann (NLPB) equation, with qualitative differences in some cases, which may be attributed to correlation effects neglected in the NLPB theory. (+info)
Sequence dependence of energy transfer in DNA oligonucleotides.
(35/1497)
The sequence, temperature, concentration, and solvent dependence of singlet energy transfer from normal DNA bases to the 2-aminopurine base in synthesized DNA oligomers were investigated by optical spectroscopy. Transfer was shown directly by a variable fluorescence excitation band at 260-280 nm. Adenine (A) is the most efficient energy donor by an order of magnitude. Stacks of A adjacent to 2AP act as an antenna for 2AP excitation. An interposed G, C, or T base between A and 2AP effectively blocks transfer from A to 2AP. Base stacking facilitates transfer, while base pairing reduces energy transfer slightly. The efficiency is differentially temperature dependent in single- and double-stranded oligomers and is highest below 0 degrees C in samples measured. An efficiency transition occurs well below the melting transition of a double-stranded decamer. The transfer efficiency in the duplex decamer d(CTGA[2AP]TTCAG)(2) is moderately dependent on the sample and salt concentration and is solvent dependent. Transfer at physiological temperature over more than a few bases is improbable, except along consecutive A's, indicating that singlet energy transfer is not a major factor in the localization of UV damage in DNA. These results have features in common with recently observed electron transfer from 2AP to G in oligonucleotides. (+info)
A unified theory of the B-Z transition of DNA in high and low concentrations of multivalent ions.
(36/1497)
We showed recently that the high-salt transition of poly[d(G-C)]. poly[d(G-C)] between B-DNA and Z-DNA (at [NaCl] = 2.25 M or [MgCl(2)] = 0.7 M) can be ascribed to the lesser electrostatic free energy of the B form, due to better immersion of the phosphates in the solution. This property was incorporated in cylindrical DNA models that were analyzed by Poisson-Boltzmann theory. The results are insensitive to details of the models, and in fair agreement with experiment. In contrast, the Z form of the poly[d(G-m5C)] duplex is stabilized by very small concentrations of magnesium. We now show that this striking difference is accommodated quantitatively by the same electrostatic theory, without any adjustable parameter. The different responses to magnesium of the methylated and nonmethylated polymers do not come from stereospecific cation-DNA interactions: they stem from an experimentally derived, modest difference in the nonelectrostatic component of the free energy difference (or NFED) between the Z and B forms. The NFED is derived from circular DNA measurements. The differences between alkaline earth and transition metal ions are explained by weak coordination of the latter. The theory also explains the induction of the transition by micromolar concentrations of cobalt hexammine, again without specific binding or adjustable parameters. Hence, in the case of the B-Z transition as in others (e.g., the folding of tRNA and of ribozymes), the effect of multivalent cations on nucleic acid structure is mediated primarily by nonspecific ion-polyelectrolyte interactions. We propose this as a general rule for which convincing counter-examples are lacking. (+info)
Multiple stress responses in Streptococcus mutans and the induction of general and stress-specific proteins.
(37/1497)
The authors have previously demonstrated that Streptococcus mutans shows an exponential-phase acid-tolerance response following an acid shock from pH 7.5 to 5.5 that enhances survival at pH 3.0. In this study the response of S. mutans H7 to acid shock was compared with the responses generated by salt, heat, oxidation and starvation. Prior induction of the acid-tolerance response did not cross-protect the cells from a subsequent challenge by the other stresses; however, prior adaptation to the other stresses, except heat (42 degrees C), protected the cells during a subsequent acid challenge at pH 3.5. Starvation by fivefold dilution of the basal medium (BM) plus fivefold reduction of its glucose content increased the numbers of survivors 12-fold, whereas elimination of glucose from fivefold-diluted BM led to a sevenfold enhancement compared to the control cells; this indicated a relationship between the acid and starvation responses. The stress responses were further characterized by comparing the 2D electrophoretic protein profiles of exponential-phase cells subjected to the various stress conditions. Cells were grown to exponential phase at pH 7.5 (37 degrees C) and then incubated for 30 min under the various stress conditions in the presence of 14C-labelled amino acids followed by cell extraction, protein separation by 2D gel electrophoresis and image analysis of the resulting autoradiograms. Using consistent twofold or greater changes in IOD % as a measure, oxidative stress resulted in the upregulation of 69 proteins, 15 of which were oxidation-specific, and in the downregulation of 24 proteins, when compared to the control cells. An acid shock from pH 7.5 to 5.5 enhanced synthesis of 64 proteins, 25 of them acid-specific, while 49 proteins exhibited diminished synthesis. The dilution of BM resulted in the increased formation of 58 proteins, with 11 starvation-specific proteins and 20 showing decreased synthesis. Some 52 and 40 proteins were enhanced by salt and heat stress, with 10 and 6 of these proteins, respectively, specific to the stress. The synthesis of a significant number of proteins was increased by more than one, but not all stress conditions; six proteins were enhanced by all five stress conditions and could be classified as general stress proteins. Clearly, the response of S. mutans to adverse environmental conditions results in complex and diverse alterations in protein synthesis to further cell survival. (+info)
The role of mismatched nucleotides in activating the hMSH2-hMSH6 molecular switch.
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We have previously shown that hMSH2-hMSH6 contains an intrinsic ATPase which is activated by mismatch-provoked ADP-->ATP exchange that coordinately induces the formation of a sliding clamp capable of hydrolysis-independent diffusion along the DNA backbone (1,2). These studies suggested that mismatch repair could be propagated by a signaling event transduced via diffusion of ATP-bound hMSH2-hMSH6 molecular switches to the DNA repair machinery. The Molecular Switch model (Fishel, R. (1998) Genes Dev. 12, 2096-2101) is considerably different than the Hydrolysis-Driven Translocation model (Blackwell, L. J., Martik, D., Bjornson, K. P., Bjornson, E. S., and Modrich, P. (1998) J. Biol. Chem. 273, 32055-32062) and makes additional testable predictions beyond the demonstration of hydrolysis-independent diffusion (Gradia, S., Subramanian, D., Wilson, T., Acharya, S., Makhov, A., Griffith, J., and Fishel, R. (1999) Mol. Cell 3, 255-261): (i) individual mismatch-provoked ADP-->ATP exchange should be unique and rate-limiting, and (ii) the k(cat x DNA) for the DNA-stimulated ATPase activity should decrease with increasing chain length. Here we have examined hMSH2-hMSH6 affinity and ATPase stimulatory activity for several DNA substrates containing mispaired nucleotides as well as the chain length dependence of a defined mismatch under physiological conditions. We find that the results are most consistent with the predictions of the Molecular Switch model. (+info)
Engineered salt-insensitive alpha-defensins with end-to-end circularized structures.
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We designed a retro-isomer and seven circularized "beta-tile" peptide analogs of a typical rabbit alpha-defensin, NP-1. The analogs retained defensin-like architecture after the characteristic end-to-end, Cys(3,31) (C I:C VI), alpha-defensin disulfide bond was replaced by a backbone peptide bond. The retro-isomer of NP-1 was as active as the parent compound, suggesting that overall topology and amphipathicity governed its antimicrobial activity. A beta-tile design with or without a single cross-bracing disulfide bond sufficed for antimicrobial activity, and some of the analogs retained activity against Escherichia coli and Salmonella typhimurium in NaCl concentrations that rendered NP-1 inactive. The new molecules had clustered positive charges resembling those in protegrins and tachyplesins, but were less cytotoxic. Such simplified alpha-defensin analogs minimize problems encountered during the oxidative folding of three-disulfide defensins. In addition, they are readily accessible to a novel thia zip cyclization procedure applicable to large unprotected peptide precursors of 31 amino acids in aqueous solutions. Collectively, these findings provide new and improved methodology to create salt-insensitive defensin-like peptides for application against bacterial diseases. (+info)
Sequence requirements for translation initiation of Rhopalosiphum padi virus ORF2.
(40/1497)
Rhopalosiphum padi virus (RhPV) is an aphid-infecting virus with a 10-kb ssRNA genome that contains two large open reading frames (ORFs). ORF1 and ORF2 encode the nonstructural and structural polyproteins, respectively. Both ORFs are preceded by noncoding regions of 500 nt that could function as internal ribosome entry segments (IRESes). The sequence for ORF2 lacks an obvious initiation codon, but an out-of-frame AUG codon is present that could translate ORF2 through a +1 frameshift. To investigate the mechanisms of translation initiation of ORF2, a series of point and deletion mutations were constructed and transcribed and translated in vitro. A bicistronic plasmid containing two copies of the RhPV intergenic region translated both ORFs efficiently, indicating that the region functioned as an IRES in vitro. Deletion analysis showed that the region required for activity of the IRES extended from 178 nt upstream and 6 nt downstream of the 5' border of ORF2. Changes in the out-of-frame AUG codon had little effect on translation initiation, but mutations that included the first and second codons of ORF2 or that disrupted a putative pseudoknot structure near the 3' end of the IRES failed to initiate protein synthesis. Sequence analysis of the in vitro synthesized proteins showed that the first amino acid of the polyprotein corresponded to the second codon of ORF2. These results show that in vitro the noncoding region upstream of RhPV ORF2 functions as an IRES that contains a pseudoknot that directs translation initiation at a non-AUG codon. If the in vitro synthesized proteins have not been processed by an aminopeptidase, translation is initiated at the second (GCA) codon of ORF2. (+info)