Structural predictions for the ligand-binding region of glycoprotein hormone receptors and the nature of hormone–receptor interactions

AbstractBackground: Glycoprotein hormones influence the development and function of the ovary, testis and thyroid by binding to specific high-affinity receptors. The extracellular domains of these receptors are members of the leucine-rich repeat (LRR) protein superfamily and are responsible for the high-affinity binding. The crystal structure of a glycoprotein hormone, namely human choriogonadotropin (hCG), is known, but neither the receptor structure, mode of hormone binding, nor mechanism for activation, have been established.Results Despite very low sequence similarity between exon-demarcated LRRs in the receptors and the LRRs of porcine ribonuclease inhibitor (RI), the secondary structures for the two repeat sets are found to be alike. Constraints on curvature and β-barrel geometry from the sequence pattern for repeated βα units suggest that the receptors contain three-dimensional structures similar to that of RI. With the RI crystal structure as a template, models were constructed for exons 2–8 of the receptors. The model for this portion of the choriogonadotropin receptor is complementary in shape and electrostatic characteristics to the surface of hCG at an identified focus of hormone–receptor interaction.Conclusion The predicted models for the structures and mode of hormone binding of the glycoprotein hormone receptors are to a large extent consistent with currently available biochemical and mutational data. Repeated sequences in β-barrel proteins are shown to have general implications for constraints on structure. Averaging techniques used here to recognize the structural motif in these receptors should also apply to other proteins with repeated sequences

Co-evolution of segregation guide DNA motifs and the FtsK translocase in bacteria: identification of the atypical Lactococcus lactis KOPS motif

Bacteria use the global bipolarization of their chromosomes into replichores to control the dynamics and segregation of their genome during the cell cycle. This involves the control of protein activities by recognition of specific short DNA motifs whose orientation along the chromosome is highly skewed. The KOPS motifs act in chromosome segregation by orienting the activity of the FtsK DNA translocase towards the terminal replichore junction. KOPS motifs have been identified in γ-Proteobacteria and in Bacillus subtilis as closely related G-rich octamers. We have identified the KOPS motif of Lactococcus lactis, a model bacteria of the Streptococcaceae family harbouring a compact and low GC% genome. This motif, 5′-GAAGAAG-3, was predicted in silico using the occurrence and skew characteristics of known KOPS motifs. We show that it is specifically recognized by L. lactis FtsK in vitro and controls its activity in vivo. L. lactis KOPS is thus an A-rich heptamer motif. Our results show that KOPS-controlled chromosome segregation is conserved in Streptococcaceae but that KOPS may show important variation in sequence and length between bacterial families. This suggests that FtsK adapts to its host genome by selecting motifs with convenient occurrence frequencies and orientation skews to orient its activity

Bioactive (3Z,5E)-11,20-Epoxybriara-3,5-dien-7,18-olide Diterpenoids from the South China Sea Gorgonian Dichotella gemmacea

Six new (3Z,5E)-11,20-epoxybriara-3,5-dien-7,18-olide diterpenoids, gemmacolides N–S (1–6), were isolated together with four known analogues, juncenolide D, and juncins R, S and U (7–10), from the South China Sea gorgonian Dichotella gemmacea. The structures of the new compounds were elucidated by the detailed analysis of spectroscopic data in combination with the comparison with reported data. The absolute configuration of 1 was determined by a TDDFT calculation of its solution ECD spectrum, affording the determination of absolute configuration of other analogues by simply comparing their ECD spectra with that of 1. The cytotoxic and antimicrobial activities of these compounds were evaluated. In preliminary in vitro bioassays, compounds 4, 5, 6, 8 and 9 showed cytotoxicity against A549 and MG63, while compounds 1, 2, 4, 7–10 showed antimicrobial activity against the fungus Septoria tritici and the bacterium Escherichia coli

Dithiooxamide Modified Glassy Carbon Electrode for the Studies of Non-Aqueous Media: Electrochemical Behaviors of Quercetin on the Electrode Surface

Electrochemical oxidation of quercetin, as an important biological molecule, has been studied in non-aqueous media using cyclic voltammetry, electrochemical impedance spectroscopy and scanning electron microscopy. To investigate the electrochemical properties of quercetin, an important flavonoid derivative, on a different surface, a new glassy carbon electrode has been developed using dithiooxamide as modifier in non-aqueous media. The surface modification of glassy carbon electrode has been performed within the 0.0 mV and +800 mV potential range with 20 cycles using 1 mM dithioxamide solution in acetonitrile. However, the modification of quercetin to both bare glassy carbon and dithiooxamide modified glassy carbon electrode surface was carried out in a wide +300 mV and +2,800 mV potential range with 10 cycles. Following the modification process, cyclic voltammetry has been used for the surface characterization in aqueous and non-aqueous media whereas electrochemical impedance spectroscopy has been used in aqueous media. Scanning electron microscopy has also been used to support the surface analysis. The obtained data from the characterization and modification studies of dithioxamide modified and quercetin grafted glassy carbon electrode showed that the developed electrode can be used for the quantitative determination of quercetin and antioxidant capacity determination as a chemical sensor electrode

Current challenges in software solutions for mass spectrometry-based quantitative proteomics

This work was in part supported by the PRIME-XS project, grant agreement number 262067, funded by the European Union seventh Framework Programme; The Netherlands Proteomics Centre, embedded in The Netherlands Genomics Initiative; The Netherlands Bioinformatics Centre; and the Centre for Biomedical Genetics (to S.C., B.B. and A.J.R.H); by NIH grants NCRR RR001614 and RR019934 (to the UCSF Mass Spectrometry Facility, director: A.L. Burlingame, P.B.); and by grants from the MRC, CR-UK, BBSRC and Barts and the London Charity (to P.C.

Significance analysis of microarray for relative quantitation of LC/MS data in proteomics

<p>Abstract</p> <p>Background</p> <p>Although fold change is a commonly used criterion in quantitative proteomics for differentiating regulated proteins, it does not provide an estimation of false positive and false negative rates that is often desirable in a large-scale quantitative proteomic analysis. We explore the possibility of applying the Significance Analysis of Microarray (SAM) method (PNAS 98:5116-5121) to a differential proteomics problem of two samples with replicates. The quantitative proteomic analysis was carried out with nanoliquid chromatography/linear iron trap-Fourier transform mass spectrometry. The biological sample model included two <it>Mycobacterium smegmatis </it>unlabeled cell cultures grown at pH 5 and pH 7. The objective was to compare the protein relative abundance between the two unlabeled cell cultures, with an emphasis on significance analysis of protein differential expression using the SAM method. Results using the SAM method are compared with those obtained by fold change and the conventional <it>t</it>-test.</p> <p>Results</p> <p>We have applied the SAM method to solve the two-sample significance analysis problem in liquid chromatography/mass spectrometry (LC/MS) based quantitative proteomics. We grew the pH5 and pH7 unlabelled cell cultures in triplicate resulting in 6 biological replicates. Each biological replicate was mixed with a common ¹⁵N-labeled reference culture cells for normalization prior to SDS/PAGE fractionation and LC/MS analysis. For each biological replicate, one center SDS/PAGE gel fraction was selected for triplicate LC/MS analysis. There were 121 proteins quantified in at least 5 of the 6 biological replicates. Of these 121 proteins, 106 were significant in differential expression by the <it>t</it>-test (<it>p </it>< 0.05) based on peptide-level replicates, 54 were significant in differential expression by SAM with Δ = 0.68 cutoff and false positive rate at 5%, and 29 were significant in differential expression by the <it>t</it>-test (<it>p </it>< 0.05) based on protein-level replicates. The results indicate that SAM appears to overcome the false positives one encounters using the peptide-based <it>t</it>-test while allowing for identification of a greater number of differentially expressed proteins than the protein-based <it>t</it>-test.</p> <p>Conclusion</p> <p>We demonstrate that the SAM method can be adapted for effective significance analysis of proteomic data. It provides much richer information about the protein differential expression profiles and is particularly useful in the estimation of false discovery rates and miss rates.</p

Computational design of syntheses leading to compound libraries or isotopically labelled targets

Although computer programs for retrosynthetic planning have shown improved and in some cases quite satisfactory performance in designing routes leading to specific, individual targets, no algorithms capable of planning syntheses of entire target libraries - important in modern drug discovery - have yet been reported. This study describes how network-search routines underlying existing retrosynthetic programs can be adapted and extended to multi-target design operating on one common search graph, benefitting from the use of common intermediates and reducing the overall synthetic cost. Implementation in the Chematica platform illustrates the usefulness of such algorithms in the syntheses of either (i) all members of a user-defined library, or (ii) the most synthetically accessible members of this library. In the latter case, algorithms are also readily adapted to the identification of the most facile syntheses of isotopically labelled targets. These examples are industrially relevant in the context of hitto-lead optimization and syntheses of isotopomers of various bioactive molecules

Biomarkers for Clinical and Incipient Tuberculosis: Performance in a TB-Endemic Country

Simple biomarkers are required to identify TB in both HIV(-)TB(+) and HIV(+)TB(+) patients. Earlier studies have identified the M. tuberculosis Malate Synthase (MS) and MPT51 as immunodominant antigens in TB patients. One goal of these investigations was to evaluate the sensitivity and specificity of anti-MS and -MPT51 antibodies as biomarkers for TB in HIV(-)TB(+) and HIV(+)TB(+) patients from a TB-endemic setting. Earlier studies also demonstrated the presence of these biomarkers during incipient subclinical TB. If these biomarkers correlate with incipient TB, their prevalence should be higher in asymptomatic HIV(+) subjects who are at a high-risk for TB. The second goal was to compare the prevalence of these biomarkers in asymptomatic, CD4(+) T cell-matched HIV(+)TB(-) subjects from India who are at high-risk for TB with similar subjects from US who are at low-risk for TB.Anti-MS and -MPT51 antibodies were assessed in sera from 480 subjects including PPD(+) or PPD(-) healthy subjects, healthy community members, and HIV(-)TB(+) and HIV(+)TB(+) patients from India. Results demonstrate high sensitivity (approximately 80%) of detection of smear-positive HIV(-)TB(+) and HIV(+)TB(+) patients, and high specificity (>97%) with PPD(+) subjects and endemic controls. While approximately 45% of the asymptomatic HIV(+)TB(-) patients at high-risk for TB tested biomarker-positive, >97% of the HIV(+)TB(-) subjects at low risk for TB tested negative. Although the current studies are hampered by lack of knowledge of the outcome, these results provide strong support for the potential of these biomarkers to detect incipient, subclinical TB in HIV(+) subjects.These biomarkers provide high sensitivity and specificity for TB diagnosis in a TB endemic setting. Their performance is not compromised by concurrent HIV infection, site of TB and absence of pulmonary manifestations in HIV(+)TB(+) patients. Results also demonstrate the potential of these biomarkers for identifying incipient subclinical TB in HIV(+)TB(-) subjects at high-risk for TB