System design and treatment efficiency of a surface flow constructed wetland receiving runoff impacted stream water.

This study reported the efficiency of a free water surface flow constructed wetland (CW) system that receives runoff impacted stream water from a forested and agricultural watershed. Investigations were conducted to examine the potential effect of hydraulic fluctuations on the CW as a result of storm events and the changes in water quality along the flow path of the CW. Based on the results, the incoming pollutant concentrations were increased during storm events and greater at the near end of the storm than at the initial time of storm. A similar trend was observed to the concentrations exiting the CW due to the wetland being a relatively small percentage of the watershed (<0.1%) that allowed delays in runoff time during storm events. The concentrations of most pollutants were significantly reduced (p < 0.05) except for nitrate (p = 0.5). Overall, this study suggests that the design of the system could feasibly function for the retention of most pollutants during storm events as the actual water quality of the outflow was significantly better by 21-71% than the inflow and the levels of pollutants were reduced to appreciable levels.

Synthesis of LiYF4, BaYF5, and NaLaF4 optical nanocrystals.

This paper reports the synthesis of high quality LiYF4, BaYF5, and NaLaF4 nanocrystals by high-temperature co-decomposition of precursors in organic solvents. Their bulk counterparts have long been used as efficient luminescent hosts for various applications including lasers, upconversion fluorescence, and quantum cutters. The particles were characterized using TEM, XRD, dynamic light scattering (DLS), and fluorescence spectrometry. Trifluoroacetic acid (CF3COOH) and the reaction temperature were crucial for the formation of NaLaF4 and LiYF4 nanoparticles. NaLaF4 was not formed without using CF3COOH, only LaF3 and NaF mixture was formed. NaLaF4 nanoparticles were obtained only when CF3COOH was added in the reaction solution and the temperature was > or =330 degrees C. For the synthesis of LiYF4,, in the absence of CF3COOH in the reaction, a mixture of YOF and LiYF4 nanoparticles was formed. Pure LiYF4 particles were obtained only until CF3COOH was added in the reaction at 340 degrees C or above. The nanoparticles were easily dispersed in organic solvents include hexane, toluene, and chloroform and formed transparent colloidal solutions. The ease of doping of these as-synthesized host nanoparticles for designed optical properties was assessed. The LiYF4, BaYF5, and NaLaF4 nanoparticles, co-doped with 20% Ytterbium (Yb) and 2% Erbium (Er), showed bright upconversion fluorescence upon 980 nm NIR excitation, confirming the high quality of as-synthesized nanoparticles. These nanoparticles are potential candidates for nano-optical devices, thin films, telecommunication, and bio-probes.

Solution structure and dynamics of yeast elongin C in complex with a von Hippel-Lindau peptide.

Elongin is a transcription elongation factor that stimulates the rate of elongation by suppressing transient pausing by RNA polymerase II at many sites along the DNA. It is heterotrimeric in mammals, consisting of elongins A, B and C subunits, and bears overall similarity to a class of E3 ubiquitin ligases known as SCF (Skp1-Cdc53 (cullin)-F-box) complexes. A subcomplex of elongins B and C is a target for negative regulation by the von Hippel-Lindau (VHL) tumor-suppressor protein. Elongin C from Saccharomyces cerevisiae, Elc1, exhibits high sequence similarity to mammalian elongin C. Using NMR spectroscopy we have determined the three-dimensional structure of Elc1 in complex with a human VHL peptide, VHL(157-171), representing the major Elc1 binding site. The bound VHL peptide is entirely helical. Elc1 utilizes two C-terminal helices and an intervening loop to form a binding groove that fits VHL(157-171). Chemical shift perturbation and dynamics analyses reveal that a global conformational change accompanies Elc1/VHL(157-171) complex formation. Moreover, the disappearance of conformational exchange phenomena on the microsecond to millisecond time scale within Elc1 upon VHL peptide binding suggests a role for slow internal motions in ligand recognition.

Latent and active p53 are identical in conformation.

p53 is a nuclear phosphoprotein that regulates cellular fate after genotoxic stress through its role as a transcriptional regulator of genes involved in cell cycle control and apoptosis. The C-terminal region of p53 is known to negatively regulate sequence specific DNA-binding of p53; modifications to the C-terminus relieve this inhibition. Two models have been proposed to explain this latency: (i) an allosteric model in which the C-terminal domain interacts with another domain of p53 or (ii) a competitive model in which the C-terminal and the core domains compete for DNA binding. We have characterized latent and active forms of dimeric p53 using gel mobility shift assays and NMR spectroscopy. We show on the basis of chemical shifts that dimeric p53 both containing and lacking the C-terminal domain are identical in conformation and that the C-terminus does not interact with other p53 domains. Similarly, NMR spectra of isolated core and tetramerization domains confirm a modular p53 architecture. The data presented here rule out an allosteric model for the regulation of p53.

Elucidation of binding determinants and functional consequences of Ras/Raf-cysteine-rich domain interactions.

Raf-1 is a critical downstream target of Ras and contains two distinct domains that bind Ras. The first Ras-binding site (RBS1) in Raf-1 has been shown to be essential for Ras-mediated translocation of Raf-1 to the plasma membrane, whereas the second site, in the Raf-1 cysteine-rich domain (Raf-CRD), has been implicated in regulating Raf kinase activity. While recognition elements that promote Ras.RBS1 complex formation have been characterized, relatively little is known about Ras/Raf-CRD interactions. In this study, we have characterized interactions important for Ras binding to the Raf-CRD. Reconciling conflicting reports, we found that these interactions are essentially independent of the guanine nucleotide bound state, but instead, are enhanced by post-translational modification of Ras. Specifically, our findings indicate that Ras farnesylation is sufficient for stable association of Ras with the Raf-CRD. Furthermore, we have also identified a Raf-CRD variant that is impaired specifically in its interactions with Ras. NMR data also suggests that residues proximal to this mutation site on the Raf-CRD form contacts with Ras. This Raf-CRD mutant impairs the ability of Ras to activate Raf kinase, thereby providing additional support that Ras interactions with the Raf-CRD are important for Ras-mediated activation of Raf-1.

Solution structure of an antimicrobial peptide buforin II.

The structure of 21-residue antimicrobial peptide buforin II has been determined by using NMR spectroscopy and restrained molecular dynamics. Buforin II adopts a flexible random structure in H2O. In trifluoroethanol (TFE)/H2O (1:1, v/v) mixture, however, buforin II assumes a regular alpha-helix between residues Val12 and Arg20 and a distorted helical structure between residues Gly7 and Pro11. The model structure obtained shows an amphipathic character in the region from Arg5 to the C-terminus, Lys21. Like other known cationic antimicrobial peptides, the amphipathic structure might be the key factor for antimicrobial activity of buforin II.

Disulfide bond formation between the n-terminal region of p56LCK and the cytoplasmic domain of CD8 studied by electrospray ionization and matrix-assisted desorption/ionization time-of-flight mass spectrometry.

Masses of the complexes formed between the C-terminal region of CD8 alpha (CD8 alpha C) and the N-terminal region of p56lck (p56lckN) were measured by using Electrospray Ionization (ESI) and Matrix-Assisted Laser Desorption/Ionization (MALDI) Mass Spectrometry (MS). The two peptides were incubated with various metal ions, Zn++, Cd++, Co++, Fe++ and Ca++. The complex formed from the incubation did not contain any metal ions. Instead, its mass suggested that the complex was formed by two disulfide bonds. The fact that the incubation of the complex with Dithiothreitol broke the complex confirmed into monomers that the complex was formed through disulfide bonds. The only disulfide-mediated complex formed was hetero-dimer (CD8 alpha C-p56lckN) and none of homo-dimers (CD8 alpha C-CD8 alpha C or p56lckN-p56lckN) were observed from ESI and MALDI-TOF mass spectrometry.

Structure of a methionine-rich segment of Escherichia coli Ffh protein.

The methionine-rich segments of the Ffh protein of Escherichia coli and its eukaryotic counterpart SRP54 are thought to bind signal sequences of secretory proteins. The structure of a chemically synthesized 25-residue-long peptide corresponding to one of the proposed methionine-rich amphiphilic helices of Ffh was determined in water and in aqueous trifluroethanol (TFE) solution using CD and NMR. An appreciable alpha-helix conformation exists even in water and this peptide assumes a stable alpha-helix along most of its length in aqueous TFE solution. It is clear that this segment of Ffh protein has a very strong propensity to form alpha-helical structure.

Structures of revertant signal sequences of Escherichia coli ribose binding protein.

Recently we reported (Yi et al., 1994) that the alpha-helical content of the signal peptide of Escherichia coli ribose binding protein, when determined by circular dichroism (CD) and two-dimensional NMR in trifluoroethanol/water solvent, is higher than that of its nonfunctional mutant signal peptide. In the present investigation, the structures of the signal peptides of two revertant ribose binding proteins in the same solvent were also determined with CD and two-dimensional 1H NMR spectroscopy. According to the CD results, both of these revertant signal peptides showed an intermediate helicity between those of wild-type and mutant signal peptides, the helical content of the revertant peptide with higher recovery of the translocation capability being higher. On the other hand, the alpha-helix regions of the wild-type and the revertant peptides as determined by NMR were shown to be the same. This discrepancy may be due to the difference in stability between identical alpha-helical stretches in wild-type and revertant peptides. A good correlation was observed between the helical content of these four ribose binding protein signal peptides in TFE/water as studied by CD and their in vivo translocation activities. It appears, therefore, that both the proper length of the helix and the stability are of functional significance.

A thermostable mutation located at the hydrophobic core of alpha 1-antitrypsin suppresses the folding defect of the Z-type variant.

A thermostable mutation, F51L, at the hydrophobic core of human alpha 1-antitrypsin (alpha 1AT) increased the conformational stability of the molecule by decreasing the unfolding rate significantly without altering the refolding rate. The mutation specifically influenced the transition between the native state and a compact intermediate, which retained approximately 70% of the far-UV CD signal, but which had most of the fluorescence signal already dequenched. The mutant alpha 1AT protein was more resistant than the wild-type protein to the insertion of the tetradecapeptide mimicking the sequence of the reactive center loop, indicating that the mutation increases the closing of the central beta-sheet, the A-sheet, in the native state. The F51L mutation enhanced the folding efficiency of the Z-type (E342K) genetic variation, which causes aggregation of the molecule in the liver. It has been shown previously that the aggregation of the Z protein occurs via loop-sheet polymerization, in which the reactive center loop of one molecule is inserted into the opening of the A-sheet of another molecule. Our results strongly suggest that the hydrophobic core of alpha 1AT regulates the opening-closing of the A-sheet and that certain genetic variations that cause opening of the A-sheet can be corrected by inserting an additional stable mutation into the hydrophobic core.

Structures of wild-type and mutant signal sequences of Escherichia coli ribose binding protein.

The structure of a chemically synthesized 25-residue-long functional signal peptide of Escherichia coli ribose binding protein was compared with that of a nonfunctional mutant-signal peptide using circular dichroism and two-dimensional 1H NMR in solvents mimicking the amphiphilic environments. The functional peptide forms an 18-residue-long alpha-helix starting from the NH2-terminal region and reaching to the hydrophobic stretch in a solvent consisting of 10% dimethylsulfoxide, 40% water, and 50% trifluoroethanol (v/v). The nonfunctional mutant peptide, which contains a Pro at position 9 instead of a Leu in the wild-type peptide, does not have any secondary structure in that solvent but forms a 12-residue-long alpha-helix within the hydrophobic stretch in water/trifluoroethanol (50:50, v/v) solvent. It seems that the Pro-9 residue in the nonfunctional peptide disturbs the helix propagation from the hydrophobic stretch to the NH2-terminal region. Because both of these peptides have stable helices within the hydrophobic stretch, it may be concluded that the additional 2 turns of the alpha-helix in the NH2-terminal region of the wild-type signal peptide is important for its function.

The conformation of glucagon in dilute aqueous solution as studied by 1H NMR.

The structure of monomeric glucagon in dilute aqueous solution was studied by 500 MHz NMR. Hydrogen-deuterium exchange experiments monitored by NMR showed that the backbone amide NHs form intrastrand hydrogen bonds suggesting the existence of some degree of compact structure. The temperature dependent shift of several amide NH resonances supported the above conclusion. The small J coupling constants arising from the interactions between 6 amide NHs and C alpha Hs (less than 6 Hz) imply a helical structure.