Summary

Scaffold proteins play pivotal roles in the regulation of signal transduction pathways by connecting upstream receptors to downstream effector molecules. During last decade, many scaffold proteins that contain caspase-recruitment domain (CARD) have been identified. Investigating the roles of CARD proteins has revealed that many play crucial roles in signaling cascades that lead to activation of nuclear factor-κB (NF-κB). In this review, we discuss the contributions of CARD proteins to NF-κB activation in various signaling cascades. In particular, we share some of our personal experiences during the initial investigation of the functions of the CARMA family of CARD proteins and then summarize the roles of these proteins in signaling pathways induced by antigen receptors, G protein-coupled receptors, receptor tyrosine kinase, and C-type lectin receptors in the context of recent progress in the field.

In an era when global biodiversity is increasingly impacted by rapidly changing climate, efforts to conserve global biodiversity may be compromised if we do not consider the uneven distribution of climate-induced threats. Here, via a novel application of an aggregate Regional Climate Change Index (RCCI) that combines changes in mean annual temperature and precipitation with changes in their interannual variability, we assess multi-dimensional climate changes across the “Global 200” ecoregions – a set of priority ecoregions designed to “achieve the goal of saving a broad diversity of the Earth’s ecosystems” – over the 21st century. Using an ensemble of 62 climate scenarios, our analyses show that, between 1991–2010 and 2081–2100, 96% of the ecoregions considered will be likely (more than 66% probability) to face moderate-to-pronounced climate changes, when compared to the magnitudes of change during the past five decades. Ecoregions at high northern latitudes are projected to experience most pronounced climate change, followed by those in the Mediterranean Basin, Amazon Basin, East Africa, and South Asia. Relatively modest RCCI signals are expected over ecoregions in Northwest South America, West Africa, and Southeast Asia, yet with considerable uncertainties. Although not indicative of climate-change impacts per se, the RCCI-based assessment can help policy-makers gain a quantitative and comprehensive overview of the unevenly distributed climate risks across the G200 ecoregions. Whether due to significant climate change signals or large uncertainties, the ecoregions highlighted in the assessment deserve special attention in more detailed impact assessments to inform effective conservation strategies under future climate change.

In this report we describe a chromatin immunoprecipitation (ChIP) protocol for two fully sequenced model diatom species Phaeodactylum tricornutum and Thalassiosira pseudonana. This protocol allows the extraction of satisfactory amounts of chromatin and gives reproducible results. We coupled the ChIP assay with real time quantitative PCR. Our results reveal that the two major histone marks H3K4me2 and H3K9me2 exist in P. tricornutum and T. pseudonana. As in other eukaryotes, H3K4me2 marks active genes whereas H3K9me2 marks transcriptionally inactive transposable elements. Unexpectedly however, T. pseudonana housekeeping genes also show a relative enrichment of H3K9me2. We also discuss optimization of the procedure, including growth conditions, cross linking and sonication. Validation of the protocol provides a set of genes and transposable elements that can be used as controls for studies using ChIP in each diatom species. This protocol can be easily adapted to other diatoms and eukaryotic phytoplankton species for genetic and biochemical studies.

The Encyclopedia of DNA Elements (ENCODE) consortium aims to identify all functional elements in the human genome including transcripts, transcriptional regulatory regions, along with their chromatin states and DNA methylation patterns. The ENCODE project generates data utilizing a variety of techniques that can enrich for regulatory regions, such as chromatin immunoprecipitation (ChIP), micrococcal nuclease (MNase) digestion and DNase I digestion, followed by deeply sequencing the resulting DNA. As part of the ENCODE project, we have developed a Web-accessible repository accessible at http://factorbook.org. In Wiki format, factorbook is a transcription factor (TF)-centric repository of all ENCODE ChIP-seq datasets on TF-binding regions, as well as the rich analysis results of these data. In the first release, factorbook contains 457 ChIP-seq datasets on 119 TFs in a number of human cell lines, the average profiles of histone modifications and nucleosome positioning around the TF-binding regions, sequence motifs enriched in the regions and the distance and orientation preferences between motif sites.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are characterized by acute respiratory failure and are associated with diverse disorders, such as pulmonary edema, pneumonia, sepsis, trauma, shock and lung contusion. Gene therapy is a potentially powerful approach to treat a variety of diseases related to ALI/ARDS. Numerous viral and non-viral methods for gene delivery to the lung have been developed, although pulmonary architecture and immune activation represent barriers to successful gene transfer. In this review, recent advances in the development of more efficient viral and non-viral gene transfer systems are discussed. In addition, the current status of gene therapy applied to ALI/ARDS-associated pulmonary diseases is reviewed. With the development of more efficient gene therapy vectors, gene therapy is a promising strategy for clinical application in the not too distant future.

An ongoing effort in the field of nanomedicine is to develop nanoplatforms with both imaging and therapeutic functions, the “nano-theranostics”. We have previously developed a human serum albumin (HSA) coated iron oxide nanoparticle (HINP) formula and used multiple imaging modalities to validate its tumor targeting attributes. In the current study, we sought to impart doxorubicin (Dox) onto the HINPs and to assess the potential of the conjugates as theranostic agents. In a typical preparation, we found that about 0.5 mg of Dox and 1 mg of iron oxide nanoparticles (IONPs, Fe content) could be loaded into 10 mg of HSA matrices. The resulting D-HINPs (Dox loaded HINPs) have a hydrodynamic size of 50 nm and are able to release Dox in a sustained fashion. More impressively, the HINPs can assist the translocation of Dox across cell membrane and even its accumulation in the nucleus. In vivo, D-HINPs retained a tumor targeting capability of HINPs, as manifested by both in vivo MRI and ex vivo immunostaining results. In a follow-up therapeutic study on a 4T1 murine breast cancer xenograft model, D-HINPs showed a striking tumor suppression effect that was comparable to Doxil and greatly outperformed free Dox. Such a strategy can be readily extended to load other types of small molecules, making HINP a promising theranostic nanoplatform.

Purpose

Tumor endothelial marker 8 (TEM8) has been reported to be upregulated in both tumor cells and tumor-associated endothelial cells in several cancer types. TEM8 antagonists and TEM8-targeted delivery of toxins have been developed as effective cancer therapeutics. The ability to image TEM8 expression would be of use in evaluating TEM8-targeted cancer therapy.

Methods

A 13-meric peptide, KYNDRLPLYISNP (QQM), identified from the small loop in domain IV of protective antigen of anthrax toxin was evaluated for TEM8 binding and labeled with 18F for small-animal PET imaging in both UM-SCC1 head-and-neck cancer and MDA-MB-435 melanoma models.

Results

A modified ELISA showed that QQM peptide bound specifically to the extracellular vWA domain of TEM8 with an IC50 value of 304 nM. Coupling 4-nitrophenyl 2-18F-fluoropropionate with QQM gave almost quantitative yield and a high specific activity (79.2±7.4 TBq/mmol, n=5) of 18F-FP-QQM at the end of synthesis. 18F-FP-QQM showed predominantly renal clearance and had significantly higher accumulation in TEM8 high-expressing UM-SCC1 tumors (2.96±0.84 %ID/g at 1 h after injection) than TEM8 low-expressing MDA-MB-435 tumors (1.38±0.56 %ID/g at 1 h after injection).

Conclusion

QQM peptide bound specifically to the extracellular domain of TEM8. 18F-FP-QQM peptide tracer would be a promising lead compound for measuring TEM8 expression. Further efforts to improve the affinity and specificity of the tracer and to increase its metabolic stability are warranted.

What determines large-scale patterns of species richness remains one of the most controversial issues in ecology. Using the distribution maps of 11 405 woody species in China, we compared the effects of habitat heterogeneity, human activities and different aspects of climate, particularly environmental energy, water–energy dynamics and winter frost, and explored how biogeographic affinities (tropical versus temperate) influence richness–climate relationships. We found that the species richness of trees, shrubs, lianas and all woody plants strongly correlated with each other, and more strongly correlated with the species richness of tropical affinity than with that of temperate affinity. The mean temperature of the coldest quarter was the strongest predictor of species richness, and its explanatory power for species richness was significantly higher for tropical affinity than for temperate affinity. These results suggest that the patterns of woody species richness mainly result from the increasing intensity of frost filtering for tropical species from the equator/lowlands towards the poles/highlands, and hence support the freezing-tolerance hypothesis. A model based on these results was developed, which explained 76–85% of species richness variation in China, and reasonably predicted the species richness of woody plants in North America and the Northern Hemisphere.

Direct or ex vivo BMP9 adenoviral gene therapy can induce massive bone formation at the injection sites and clearly promote spinal fusion. A comprehensive analysis of the osteogenic activity indicated that BMP9 was one of the most potent inducers of osteogenic differentiation both in vitro and in vivo among 14 types of human BMPs. However, genetic variations and whether they correlated with OPLL were not considered. We have sequenced the complete BMP9 gene in 450 patients with OPLL and in 550 matched controls. Analyses were performed on single markers and haplotypes. Single marker tests identified 6 SNPs, among which the minor alleles of rs7923671 (T>C; P = 0.0026; OR: 1.33, CI: 1.10–1.60), rs75024165 (C>T, Thr304Met; P<0.001; OR: 1.76, CI: 1.47–2.12) and rs34379100 (A>C; P<0.001; OR: 1.52, CI: 1.27–1.82) were associated with OPLL. Logistic regression analysis showed that the additive model of rs75024165 (TT vs. CT vs. CC; P<0.001; OR: 1.74) and rs34379100 (CC vs. AC vs. AA; P = 0.003; OR: 1.95) retained statistical significance when adjusted for clinical and demographic characteristics. Linkage disequilibrium (LD) analysis identified one 3 kb block of intense LD in BMP9 and one specific haplotype, CTCA (P<0.001; OR: 2.37), that contained the OPLL-associated risk alleles and was a risk factor for OPLL. This haplotype is associated with increased severity of OPLL, as shown by the distribution of ossified vertebrae in patients with OPLL (P = 0.001). In summary, in the Chinese population studied, SNPs in the BMP9 gene appear to contribute to the risk of OPLL in association with certain clinical and demographic characteristics. The severity of OPLL seems to be mediated predominantly by genetic variations in a 3kb BMP9 locus with the specific haplotype CTCA.

Ceramidase plays an important role in regulating the metabolism of sphingolipids, such as ceramide, sphingosine (SPH), and sphingosine-1-phosphate (S1P), by controlling the hydrolysis of ceramide. Here we report the cloning and biochemical characterization of a neutral ceramidase from the red flour beetle Tribolium castaneum which is an important storage pest. The Tribolium castaneum neutral ceramidase (Tncer) is a protein of 696 amino acids. It shares a high degree of similarity in protein sequence to neutral ceramidases from various species. Tncer mRNA levels are higher in the adult stage than in pre-adult stages, and they are higher in the reproductive organs than in head, thorax, and midgut. The mature ovary has higher mRNA levels than the immature ovary. Tncer is localized to the plasma membrane. It uses various ceramides (D-erythro-C6, C12, C16, C18:1, and C24:1-ceramide) as substrates and has an abroad pH optimum for its in vitro activity. Tncer has an optimal temperature of 37 °C for its in vitro activity. Its activity is inhibited by Fe2+. These results suggest that Tncer has distinct biochemical properties from neutral ceramidases from other species.

Alkaline phosphatase (AP) is a key enzyme for phytoplankton to utilize dissolved organic phosphorus (DOP) when dissolved inorganic phosphorus is limited. While three major types of AP and their correspondingly diverse subcellular localization have been recognized in bacteria, little is known about AP in eukaryotic phytoplankton such as dinoflagellates. Here, we isolated a full-length AP cDNA from a latest-diverging dinoflagellate genus Alexandrium, and conducted comparative analyses with homologs from a relatively basal (Amphidinium carterae) and late-diverging (Karenia brevis) lineage of dinoflagellates as well as other eukaryotic algae. New data and previous studies indicate that AP is common in dinoflagellates and most other major eukaryotic groups of phytoplankton. AP sequences are more variable than many other genes studied in dinoflagellates, and are divergent among different eukaryotic phytoplankton lineages. Sequence comparison to the other characterized APs suggests that dinoflagellates and some other eukaryotic phytoplankton possess the putative AP as phoA type, but some other eukaryotic phytoplankton seem to have other types. Phylogenetic analyses based on AP amino acid sequences indicated that the “red-type” eukaryotic lineages formed a monophyletic group, suggesting a common origin of their APs. As different amino acid sequences have been found to predictably determine different spatial distribution in the cells, which may facilitate access to different pools of DOP, existing computational models were adopted to predict the subcellular localizations of putative AP in the three dinoflagellates and other eukaryotic phytoplankton. Results showed different subcellular localizations of APs in different dinoflagellates and other lineages. The linkage between AP sequence divergence, subcellular localization, and ecological niche differentiation requires rigorous experimental verification, and this study now provides a framework for such a future effort.

Herein we report a new type of in vivo fluorogenic probe that enables simultaneous and active targeting of overexpressed receptors, αvβ3 integrins, and extracellular proteases, matrix metalloproteinases (MMPs), in the tumor regions. This c(RGDyK)-conjugated MMP fluorogenic probe efficiently targets the tumor regions with high retention time while maintaining receptor binding affinity and substrate activity. The probe minimizes nonspecific accumulation, and thus demonstrating improved tumor-to-background signal ratio (T/N) in both αvβ3 integrin- and MMP-overexpressing U87MG tumor-bearing mouse model. This strategy can be easily tuned for a wide array of applications targeting various receptors and extracellular proteases in vivo.

Rationale: The differentiation of fibroblasts into myofibroblasts is a cardinal feature of idiopathic pulmonary fibrosis (IPF). The transcription factor Yin Yang 1 (YY1) plays a role in the proliferation and differentiation of diverse cell types, but its role in fibrotic lung diseases is not known.

Objectives: To elucidate the mechanism by which YY1 regulates fibroblast differentiation and lung fibrosis.

Methods: Lung fibroblasts were cultured with transforming growth factor (TGF)-β or tumor necrosis factor-α. Nuclear factor (NF)-κB, YY1, and α-smooth muscle actin (SMA) were determined in protein, mRNA, and promoter reporter level. Lung fibroblasts and lung fibrosis were assessed in a partial YY1-deficient mouse and a YY1f/f conditional knockout mouse after being exposed to silica or bleomycin.

Measurements and Main Results: TGF-β and tumor necrosis factor-α up-regulated YY1 expression in lung fibroblasts. TGF-β–induced YY1 expression was dramatically decreased by an inhibitor of NF-κB, which blocked I-κB degradation. YY1 is significantly overexpressed in both human IPF and murine models of lung fibrosis, including in the aggregated pulmonary fibroblasts of fibrotic foci. Furthermore, the mechanism of fibrogenesis is that YY1 can up-regulate α-SMA expression in pulmonary fibroblasts. YY1-deficient (YY1+/−) mice were significantly protected from lung fibrosis, which was associated with attenuated α-SMA and collagen expression. Finally, decreasing YY1 expression through instilled adenovirus-cre in floxed-YY1f/f mice reduced lung fibrosis.

Conclusions: YY1 is overexpressed in fibroblasts in both human IPF and murine models in a NF-κB–dependent manner, and YY1 regulates fibrogenesis at least in part by increasing α-SMA and collagen expression. Decreasing YY1 expression may provide a new therapeutic strategy for pulmonary fibrosis.

We and others have recently proposed the synthesis of composite nanoparticles that offer strongly enhanced functionality. Here we have used a flower-shaped Au-Fe3O4 nanoparticle as a template to construct an optical probe containing Cy5.5-GPLGVRG-TDOPA on the iron oxide surface and SH-PEG5000 on the gold surface that can be specifically activated by matrix metalloproteinases (MMPs) expressed in tumors. Gold nanoparticles have excellent quenching properties, but labile surface chemistry in vivo; on the other hand, iron oxide nanoparticles afford robust surface chemistry, but are suboptimal as energy receptors. By a marriage of the two, we have produced a unified structure with performance that is unachievable with the separate components. Our results are a further demonstration that the architecture of nanoparticles can be modulated to tailor their function as molecular imaging/therapeutic agents.

Background

The Chinese white wax scale, Ericerus pela Chavannes is economically significant for its role in wax production. This insect has been bred in China for over a thousand years. The wax secreted by the male scale insect during the second-instar larval stage has been widespread used in wax candle production, wax printing, engraving, Chinese medicine, and more recently in the chemical, pharmaceutical, food, and cosmetics industries. However, little is known about the mechanisms responsible for white wax biosynthesis. The characterization of its larval transcriptome may promote better understanding of wax biosynthesis.

Methodology/Principal Findings

In this study, characterization of the transcriptome of E. pela during peak wax secretion was performed using Illumina sequencing technology. Illumina sequencing produced 41,839 unigenes. These unigenes were annotated by blastx alignment against the NCBI Non-Redundant (NR), Swiss-Prot, KEGG, and COG databases. A total of 104 unigenes related to white wax biosynthesis were identified, and 15 of them were selected for quantitative real-time PCR analysis. We evaluated the variations in gene expression across different development stages, including egg, first/second instar larvae, male pupae, and male and female adults. Then we identified five genes involved in white wax biosynthesis. These genes were expressed most strongly during the second-instar larval stage of male E. pela.

Conclusion/Significance

The transcriptome analysis of E. pela during peak wax secretion provided an overview of gene expression information at the transcriptional level and a resource for gene mining. Five genes related to white wax biosynthesis were identified.

EGF activates NF-κB and constitutively activated NF-κB contributes to EGFR mutation-associated tumorigenesis, but it remains unclear precisely how EGFR signaling leads to NF-κB activation. Here we report that CARMA3, a Caspase Recruitment Domain (CARD)-containing scaffold molecule, is required for EGF-induced NF-κB activation. CARMA3 deficiency impaired the activation of the IKK complex following EGF stimulation, resulting in a defect of EGF-induced IκBα phosphorylation and NF-κB activation. We found that CARMA3 and Bcl10 contributed to several characteristics of EGFR-associated malignancy, including proliferation, survival, migration, and invasion. Most importantly, CARMA3 contributed to tumor growth in vivo. Our findings elucidate a crucial link between EGFR-proximal signaling components and the downstream IKK complex, and they suggest a new therapeutic target for treatment of EGFR-driven cancers.

Nanomaterials provide large surface areas, relative to their volumes, on which to load functions. One challenge, however, has been to achieve precise control in loading multiple functionalities. Traditional bioconjugation techniques, which randomly target the surface functional groups of nanomaterials, have been found increasingly inadequate for such control--a drawback which may substantially slow down or prohibit the translational efforts. In the current study, we evaluated ferritin nanocages as candidate nanoplatforms for multifunctional loading. Ferritin nanocages can be either genetically or chemically modified to impart functionalities to their surfaces, and metal cations can be encapsulated in their interiors by association with metal binding sites. Moreover, different types of ferritin nanocages can be disassembled under acidic condition and reassembled at pH of 7.4, providing a facile way to achieve function hybridization. We were able to use combinations of these unique properties to produce a number of multifunctional ferritin nanostructures with precise control of their composition. We then studied these nanoparticles, both in vitro and in vivo, to evaluate their potential suitability as multimodality imaging probes. A good tumor targeting profile was observed, which was attributable to both the enhanced permeability and retention (EPR) effect and biovector mediated targeting. This, in combination with the generalizability of the function loading techniques, promises ferritin particles as a powerful nanoplatfom in the era of nanomedicine.

The NF-κB family of transcription factors plays a crucial role in cell activation, survival and proliferation. Its aberrant activity results in cancer, immunodeficiency or autoimmune disorders. Over the past two decades, tremendous progress has been made in our understanding of the signals that regulate NF-κB activation, especially how scaffold proteins link different receptors to the NF-κB-activating complex, the IκB kinase complex. The growing number of these scaffolds underscores the complexity of the signaling networks in different cell types. In this review, we discuss the role of scaffold molecules in signaling cascades induced by stimulation of antigen receptors, G-protein-coupled receptors and C-type Lectin receptors, resulting in NF-κB activation. Especially, we focus on the family of Caspase recruitment domain (CARD)-containing proteins known as CARMA and their function in activation of NF-κB, as well as the link of these scaffolds to the development of various neoplastic diseases through regulation of NF-κB.

The conjunctival microcirculation in 14 pediatric and 8 adult sickle cell anemia (SCA) patients was studied using computer-assisted intravital microscopy. The bulbar conjunctiva in SCA patients in both age groups exhibited a blanched/avascular appearance characterized by decreased vascularity. SCA patients from both age groups had many of the same abnormal morphometric {vessel diameter, vessel distribution, morphometry (shape), tortuosity, arteriole:venule (A:V) ratio, and hemosiderin deposits} and dynamic {vessel sludging/sludged flow, boxcar blood (trickled) flow and abnormal flow velocity} abnormalities. A severity index (SI) was computed to quantify the degree of vasculopathy for comparison between groups. The severity of vasculopathy differed significantly between the pediatric and adult patients (SI: 4.2 ± 1.8 vs 6.6 ± 2.4; p=0.028), indicative of a lesser degree of overall severity in the pediatric patients. Specific abnormalities that were less prominent in the pediatric patients included abnormal vessel morphometry and tortuosity. Sludged flow, abnormal vessel distribution, abnormal A:V ratio, and boxcar flow, appeared in high prevalence in both age groups. The results indicate that SCA microvascular abnormalities develop in childhood and the severity of vasculopathy likely progresses with age. Intervention and effective treatment/management modalities should target pediatric patients to ameliorate, slow down or prevent progressive microvascular deterioration.

Mammalian tooth development relies heavily on the reciprocal and sequential interactions between cranial neural crest-derived mesenchymal cells and stomadial epithelium. During mouse tooth development, odontogenic potential, that is, the capability to direct an adjacent tissue to form a tooth, resides in dental epithelium initially, and shifts subsequently to dental mesenchyme. Recent studies have shown that mouse embryonic dental epithelium possessing odontogenic potential is able to induce the formation of a bioengineered tooth crown when confronted with postnatal mesenchymal stem cells of various sources. Despite many attempts, however, postnatal stem cells have not been used successfully as the epithelial component in the generation of a bioengineered tooth. We show here that epithelial sheets of cultured human keratinocytes, when recombined with mouse embryonic dental mesenchyme, are able to support tooth formation. Most significantly, human keratinocytes, recombined with mouse embryonic dental mesenchyme in the presence of exogenous FGF8, are induced to express the dental epithelial marker PITX2 and differentiate into enamel-secreting ameloblasts that develop a human-mouse chimeric whole tooth crown. We conclude that in the presence of appropriate odontogenic signals, human keratinocytes can be induced to become odontogenic competent; and that these are capable of participating in tooth crown morphogenesis and differentiating into ameloblasts. Our studies identify human keratinocytes as a potential cell source for in vitro generation of bioengineered teeth that may be used in replacement therapy.

In the crystal structure of the centrosymmetric binuclear title complex, [Ni2(C12H8N2O2S)2(CH3OH)4], there are inter­molecular O—H⋯O, O—H⋯N and O—H⋯S hydrogen bonds. These help to stabilize the structure and link the mol­ecules, forming a three-dimensional network structure. The Ni2+ cation exists in a slightly distorted octahedral NiNO5 coordination environment. The thio­phene rings are disordered over two equivalent conformations with occupancies of 0.881 (3) and 0.119 (3).

T cell activation is tightly regulated to avoid autoimmunity. Gene related to anergy in lymphocytes (GRAIL, encoded by Rnf128) is an E3 ubiquitin ligase associated with T cell tolerance. Here we generated and analyzed GRAIL-deficient mice and found they were resistant to immune tolerance induction and exhibited greater susceptibility to autoimmune diseases than wild-type mice. GRAIL-deficient naïve T cells, after activation, exhibited increased proliferation and cytokine expression than controls and did not depend on costimulation for effector generation. Moreover, GRAIL-deficient regulatory T (Treg) cells displayed reduced suppressive function, associated with increased Th17 cell-related gene expression. GRAIL-deficient naïve and Treg cells were less efficient in downregulating T cell receptor (TCR)-CD3 expression after activation, and exhibited increased NFATc1 transcription factor expression; GRAIL expression promoted CD3 ubiquitinylation. Our results indicate that GRAIL, by mediating TCR-CD3 degradation, regulates naïve T cell tolerance induction and Treg cell function.

The X-ray crystal structure of recombinant epi-isozizaene synthase (EIZS), a sesquiterpene cyclase from Streptomyces coelicolor A3(2), has been determined at 1.60 Å resolution. Specifically, the structure of wild-type EIZS is that of its closed conformation in complex with 3 Mg2+ ions, inorganic pyrophosphate (PPi), and the benzyltriethylammonium cation (BTAC). Additionally, the structure of D99N EIZS has been determined in an open, ligand-free conformation at 1.90 Å resolution. Comparison of these two structures provides the first view of conformational changes required for substrate binding and catalysis in a bacterial terpenoid cyclase. Moreover, the binding interactions of BTAC may mimic those of a carbocation intermediate in catalysis. Accordingly, the aromatic rings of F95, F96, and F198 appear well-oriented to stabilize carbocation intermediates in the cyclization cascade through cation-π interactions. Mutagenesis of aromatic residues in the enzyme active site results in the production of alternative sesquiterpene product arrays due to altered modes of stabilization of carbocation intermediates as well as altered templates for the cyclization of farnesyl diphosphate. Accordingly, the 1.64 Å resolution crystal structure of F198A EIZS complexed with 3 Mg2+ ions, PPi, and BTAC reveals an alternative binding orientation of BTAC; alternative binding orientations of a carbocation intermediate could lead to the formation of alternative products. Finally, the crystal structure of wild-type EIZS complexed with 4 Hg2+ ions has been determined at 1.90 Å resolution, showing that metal binding triggers a significant conformational change of helix G to cap the active site.

Here we describe the design, preparation and characterization of 10 EF-Tu mutants of potential utility for the study of Escherichia coli elongation factor Tu (EF-Tu) interaction with tRNA by a fluorescence resonance energy transfer assay. Each mutant contains a single cysteine residue at positions in EF-Tu that are proximal to tRNA sites within the aminoacyl-tRNA·EF-Tu·GTP ternary complex that have previously been labeled with fluorophores. These positions fall in the 323–326 and 344–348 regions of EF-Tu, and at the C terminus. The EF-Tus were isolated as N-terminal fusions to glutathione S-transferase (GST), which was cleaved to yield intact EF-Tus. The mutant EF-Tus were tested for binding to GDP, binding to tRNA in gel retardation and protection assays, and activity in poly-U translation in vitro. The results indicate that at least three EF-Tu mutants, K324C, G325C and E348C, are suitable for further studies. Remarkably, GST fusions that were not cleaved were also active in the various assays, despite the N-terminal fusion.

We report the development of CoMFA analysis models that correlate the 3D chemical structures of 80 compounds with 6-5 fused ring system synthesized in our laboratory and their inhibitory potencies against tgDHFR and rlDHFR. In addition to conventional CoMFA analysis, we used two routines available in the literature aimed at the optimization of CoMFA: all-orientation search (AOS) and cross-validated r2-guided region selection (q2-GRS) to further optimize the models. During this process, we identified a problem associated with q2-GRS routine and modified using two strategies. Thus, for the inhibitory activity against each enzyme (tgDHFR and rlDHFR), five CoMFA models were developed using the conventional CoMFA, AOS optimized CoMFA, the original q2-GRS optimized CoMFA and the modified q2-GRS optimized CoMFA using the first and the second strategy. In this study, we demonstrate that the modified q2-GRS routines are superior to the original routine. On the basis of the steric contour maps of the models, we designed four new compounds in the 2,4-diamino-5-methyl-6-phenylsulfanyl-substituted pyrrolo[2,3-d]pyrimidine series. As predicted, the new compounds were potent and selective inhibitors of tgDHFR. One of them, 2,4-Diamino-5-methyl-6-(2′,6′-dimethylphenylthio)pyrrolo[2,3-d]pyrimidine, is the first 6-5 fused ring system compound with nanomolar tgDHFR inhibitory activity. The HCl salt of this compound was also prepared to increase solubity. Both forms of the drug were tested in vivo in a T. gondii infection mouse model. The results indicate that both forms were active with the HCl salt significantly more potent than the free base.