Homodimeric hexaprenyl pyrophosphate synthase from the thermoacidophilic crenarchaeon Sulfolobus solfataricus displays asymmetric subunit structures

Hexaprenyl pyrophosphate synthase (HexPPs) from Sulfolobus solfataricus catalyzes the synthesis of trans-C(30)-hexaprenyl pyrophosphate (HexPP) by reacting two isopentenyl pyrophosphate molecules with one geranylgeranyl pyrophosphate. The crystal structure of the homodimeric C(30)-HexPPs resembles those of other trans-prenyltransferases, including farnesyl pyrophosphate synthase (FPPs) and octaprenyl pyrophosphate synthase (OPPs). In both subunits, 10 core helices are arranged about a central active site cavity. Leu164 in the middle of the cavity controls the product chain length. Two protein conformers are observed in the S. solfataricus HexPPs structure, and the major difference between them occurs in the flexible region of residues 84 to 100. Several helices (alphaI, alphaJ, alphaK, and part of alphaH) and the associated loops have high-temperature factors in one monomer, which may be related to the domain motion that controls the entrance to the active site. Different side chain conformations of Trp136 in two HexPPs subunits result in weaker hydrophobic interactions at the dimer interface, in contrast to the symmetric pi-pi stacking interactions of aromatic side chains found in FPPs and OPPs. Finally, the three-conformer switched model may explain the catalytic process for HexPPs.     

Identification and characterization of NeuB3 from Campylobacter jejuni as a pseudaminic acid synthase

Campylobacter jejuni and Campylobacter coli are the main causes of bacterial diarrhea worldwide, and Helicobacter pylori is known to cause duodenal ulcers. In all of these pathogenic organisms, the flagellin proteins are heavily glycosylated with a 2-keto-3-deoxy acid, pseudaminic acid (5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid). The presence of pseudaminic acid is required for the proper development of the flagella and is thereby necessary for motility in, and invasion of, the host. In this study we report the first characterization of NeuB3 from C. jejuni as a pseudaminic acid synthase; the enzyme directly responsible for the biosynthesis of pseudaminic acid. Pseudaminic acid synthase catalyzes the condensation of phosphoenolpyruvate (PEP) with the hexose, 2,4-diacetamido-2,4,6-trideoxy-L-altrose (6-deoxy-AltdiNAc), to form pseudaminic acid and phosphate. The enzymatic activity was monitored using 1H and 31P NMR spectroscopy, and the product was isolated and characterized. Kinetic analysis reveals that pseudaminic acid synthase requires the presence of a divalent metal ion for catalysis and that optimal catalysis occurs at pH 7.0. A coupled enzymatic assay gave the values for k(cat) of 0.65 +/- 0.01 s(-1), K(m)PEP of 6.5 +/- 0.4 microM, and K(m)6-deoxy-AltdiNAc of 9.5 +/- 0.7 microM. A mechanistic study on pseudaminic acid synthase, using [2-18O]PEP, shows that catalysis proceeds through a C-O bond cleavage mechanism similar to other PEP condensing synthases such as sialic acid synthase.     

Wilms' tumor 1 (WT1) gene in hematopoiesis: a surrogate marker of cell proliferation as a possible mechanism of action?

BACKGROUND: Wilms' tumor 1 (WT1) gene expression is seen in a significant number of cases of human neoplasia; however, the mechanism of action remains to be clarified. We hypothesized that WT1 gene is a surrogate marker of proliferation in normal hematopoietic cells and leukemias. While we and others have recognized its value as a tool for the detection of minimal residual disease (MRD), the objective of this study was to confirm our hypothesis regarding normal. METHODS: Samples from healthy donors (n=16) and UC blood (n=9) were cultured in Methocult for 21 days. Colonies were analyzed on days 7, 14 and 21 by RT-PCR for WT1 gene expression. Our positive controls were samples from patients with leukemia (n=91). Negative controls were from normal volunteers without stimulation (n=26). RESULTS: Results showed a statistically significant difference (P<0.0001) between cultured groups, with the highest level of WT1 gene expression in the positive controls and on day 14, when cells are at their maximal proliferation. DISCUSSION: In conclusion, WT1 gene expression in the proliferating colonies was highest on day 14, although less than in leukemia samples, confirming our hypothesis that WT1 gene is a surrogate marker of proliferation, not only in leukemogenesis but also, to a lesser degree, in normal cell proliferation.     

Persistence of lung inflammation and lung cytokines with high-resolution CT abnormalities during recovery from SARS

Background

During the acute phase of severe acute respiratory syndrome (SARS), mononuclear cells infiltration, alveolar cell desquamation and hyaline membrane formation have been described, together with dysregulation of plasma cytokine levels. Persistent high-resolution computed tomography (HRCT) abnormalities occur in SARS patients up to 40 days after recovery.

Methods

To determine further the time course of recovery of lung inflammation, we investigated the HRCT and inflammatory profiles, and coronavirus persistence in bronchoalveolar lavage fluid (BALF) of 12 patients at recovery at 60 and 90 days.

Results

At 60 days, compared to normal controls, SARS patients had increased cellularity of BALF with increased alveolar macrophages (AM) and CD8 cells. HRCT scores were increased and correlated with T-cell numbers and their subpopulations, and inversely with CD4/CD8 ratio. TNF-α, IL-6, IL-8, RANTES and MCP-1 levels were increased. Viral particles in AM were detected by electron microscopy in 7 of 12 SARS patients with high HRCT score. On day 90, HRCT scores improved significantly in 10 of 12 patients, with normalization of BALF cell counts in 6 of 12 patients with repeat bronchoscopy. Pulse steroid therapy and prolonged fever were two independent factors associated with delayed resolution of pneumonitis, in this non-randomized, retrospective analysis.

Conclusion

Resolution of pneumonitis is delayed in some patients during SARS recovery and may be associated with delayed clearance of coronavirus, Complete resolution may occur by 90 days or later.     

Remediation of Metal-Contaminated Soil by an Integrated Soil Washing-Electrolysis Process

Chelating agents such as EDTA and DTPA are often used to remove metals from soil. However, their toxicity, bio-recalcitrance, and problems with recovery of heavy metal and chelating agents severely limit their applications. A biodegradable chelating agent, LED3A, and two surfactants, SDS and Triton X 100, were evaluated as potential alternatives for remediation of metal-contaminated soil.

LED3A alone only removed 40% of cadmium the addition of surfactant significantly enhanced its cadmium removal capacity up to 80% for a wide range of pH (5 to 11). The enhancement increased with both surfactant concentrations and LED3A concentrations. Because LED3A had a much higher removal capacity for copper, the synergistic effect of surfactant-LED3A mixture was less obvious. Sequential extraction analysis indicated that the LED3A not only removed copper from carbonate and Fe-Mn oxide fraction, but also from organic fractions. A three-dimension electrolysis reactor could effectively recover both metals and LED3A-SDS within thirty minutes. The combined soil washing by LED3A-surfactants and electrolysis provides a potential approach for remediation of copper- and cadmium-contaminated soils.     

Predicting enzyme subclass by functional domain composition and pseudo amino acid composition

As a continuous effort to use the sequence approach to identify enzymatic function at a deeper level, investigations are extended from the main enzyme classes (Protein Sci. 2004, 13, 2857-2863) to their subclasses. This is indispensable if we wish to understand the molecular mechanism of an enzyme at a deeper level. For each of the 6 main enzyme classes (i.e., oxidoreductase, transferase, hydrolase, lyase, isomerase, and ligase), a subclass training dataset is constructed. To reduce homologous bias, a stringent cutoff was imposed that all the entries included in the datasets have less than 40% sequence identity to each other. To catch the core feature that is intimately related to the biological function, the sample of a protein is represented by hybridizing the functional domain composition and pseudo amino acid composition. On the basis of such a hybridization representation, the FunD-PseAA predictor is established. It is demonstrated by the jackknife cross-validation tests that the overall success rate in identifying the 21 subclasses of oxidoreductases is above 86%, and the corresponding rates in identifying the subclasses of the other 5 main enzyme classes are 94-97%. The high success rates imply that the FunD-PseAA predictor may become a useful tool in bioinformatics and proteomics of the post-genomic era.     

Coupling interaction between thromboxane A2 receptor and alpha-13 subunit of guanine nucleotide-binding protein

G protein-coupled receptors (GPCRs) form a large superfamily of membrane proteins that play an essential role in modulating many vital physiological events, such as cell communication, neurotransmission, sensory perception, and chemotaxis. Understanding of the 3D (dimensional) structures of these receptors and their binding interactions with G proteins will help in the design of drugs for the treatment of GPCR-related diseases. By means of the approach of structural bioinformatics, the 3D structures of human alpha-13 subunit of guanine nucleotide-binding protein (G alpha 13) and human thromboxane A2 (TXA2) receptor were developed. The former plays an important role in the control of cell growth that may serve as a prototypical G protein; the latter is a target for nitric oxide-mediated desensitization that may serve as a prototypical GPCR. On the basis of the 3D models, their coupling interactions were investigated via docking studies. It has been found that the two proteins are coupled with each other mainly through the interaction between the minigene of G alpha 13 and the 3rd intracellular loop of the TXA2 receptor, consistent with the existing deduction in the literatures. However, it has also been observed via a close view that some residues of the TXA2 receptor that are sequentially far away but spatially quite close to the loop region are also involved in forming hydrogen bonds with the minigene of G alpha 13. These findings may provide useful information for conducting mutagenesis and reveal the molecular mechanism how the human TXA2 receptor interact with G alpha 13 to activate intracellular signaling. The findings may also provide useful insights for stimulating new therapeutic approaches by manipulating the interaction of the receptor with the relevant G proteins.     

Functional analysis of all nonribosomal peptide synthetases in Cochliobolus heterostrophus reveals a factor, NPS6, involved in virulence and resistance to oxidative stress

Nonribosomal peptides, made by nonribosomal peptide synthetases, have diverse biological activities, including roles as fungal virulence effectors. Inspection of the genome of Cochliobolus heterostrophus, a fungal pathogen of maize and a member of a genus noted for secondary metabolite production, revealed eight multimodular nonribosomal peptide synthase (NPS) genes and three monomodular NPS-like genes, one of which encodes a nonribosomal peptide synthetase/polyketide synthase hybrid enzyme presumed to be involved in synthesis of a peptide/polyketide molecule. Deletion of each NPS gene and phenotypic analyses showed that the product of only one of these genes, NPS6, is required for normal virulence on maize. NPS6 is also required for resistance to hydrogen peroxide, suggesting it may protect the fungus from oxidative stress. This and all other nps mutants had normal growth, mating ability, and appressoria. Real-time PCR analysis showed that expression of all NPS genes is low (relative to that of actin), that all (except possibly NPS2) are expressed during vegetative growth, and that expression is induced by nitrogen starvation. Only NPS6 is unfailingly conserved among euascomycete fungi, including plant and human pathogens and saprobes, suggesting the possibility that NPS6 activity provides oxidative stress protection during both saprobic and parasitic growth.     

Specific secondary structures in the capsid-coding region of giardiavirus transcript are required for its translation in Giardia lamblia

Enhanced translation of giardiavirus (GLV)-luciferase chimeric mRNA in Giardia lamblia requires the presence of the initial 264 nucleotides of the viral capsid-coding region. A 13 nt downstream box (DB) sequence within this region, complementary to a 15 nt sequence near the 3' end of G. lamblia 16 S-like ribosomal RNA (rRNA), was found to be essential for the enhanced translation. However, DB is located 64-78 nt downstream of the initiation codon, whereas an exponential increase of translation efficiency depends on a further increment of the coding region from nucleotides 111 to 264. Thus, there could be additional structural requirements for translation enhancement in the region downstream from DB. Four major stem-loop structures, designated I to IV, were identified in the MFOLD-predicted secondary structure of the 264 nt capsid-coding region with an estimated minimum free energy (DeltaG degrees ) of -77.16 kcal x mol(-1). Our chemical probing analysis of the free 264 nt RNA molecule in solution supports the predicted presence of stem-loops I, II and III, but casts doubts on stem-loop IV. It suggests, instead, the presence of a stem-loop IVA at a nearby location in the molecule. Site-directed mutagenesis designed to disrupt stem-loop structures I, II, III or IVA resulted in drastic reduction of translation efficiency, which was restored by compensatory sequence changes to regenerate individual stem-loop structures. Mutations disrupting the originally designated stem-loop IV did not exert any detectable effect on translation. However, alterations of the sequence UCUCC between nucleotides 216 and 220 in the flexible loop region of the revised secondary structure led to a precipitous drop in translation. Another stem-loop predicted by MFOLD that consists of a major portion of the DB sequence was examined by chemical probing but found little experimental support. Changes of the DB sequence without affecting the postulated stem structure led to drastic losses of translation efficiency. Thus, a simple structural basis for the enhanced translation could be that stem-loops I, II, III and IVA and the UCUCC sequence may facilitate the interaction between DB and the anti-DB in 16 S-like rRNA in initiating translation of GLV mRNA in G. lamblia.