The selectivity of tyrosine 280 of human 11beta-hydroxysteroid dehydrogenase type 1 in inhibitor binding

11beta-Hydroxysteroid dehydrogenase type 1 is a homodimer where the carboxyl terminus of one subunit covers the active site of the dimer partner. Based on the crystal structure with CHAPS, the carboxyl terminal tyrosine 280 (Y280) has been postulated to interact with the substrate/inhibitor at the binding pocket of the dimer partner. However, the co-crystal structure with carbenoxolone argues against this role. To clarify and reconcile these findings, here we report our mutagenesis data and demonstrate that Y280 is not involved in substrate binding but rather plays a selective role in inhibitor binding. The involvement of Y280 in inhibitor binding depends on the inhibitor chemical structure. While Y280 is not involved in the binding of carbenoxolone, it is critical for the binding of glycyrrhetinic acid.     

Single Oral Dose Pharmacokinetics of Decursin and Decursinol Angelate in Healthy Adult Men and Women

The ethanol extract of Angelica gigas Nakai (AGN) root has promising anti-cancer and other bioactivities in rodent models. It is currently believed that the pyranocoumarin isomers decursin (D) and decursinol angelate (DA) contribute to these activities. We and others have documented that D and DA were rapidly converted to decursinol (DOH) in rodents. However, our in vitro metabolism studies suggested that D and DA might be metabolized differently in humans. To test this hypothesis and address a key question for human translatability of animal model studies of D and DA or AGN extract, we conducted a single oral dose human pharmacokinetic study of D and DA delivered through an AGN-based dietary supplement Cogni.Q (purchased from Quality of Life Labs, Purchase, NY) in twenty healthy subjects, i.e., 10 men and 10 women, each consuming 119 mg D and 77 mg DA from 4 vegicaps. Analyses of plasma samples using UHPLC-MS/MS showed mean time to peak concentration (T_max) of 2.1, 2.4 and 3.3 h and mean peak concentration (C_max) of 5.3, 48.1 and 2,480 nmol/L for D, DA and DOH, respectively. The terminal elimination half-life (t_1/2) for D and DA was similar (17.4 and 19.3 h) and each was much longer than that of DOH (7.4 h). The mean area under the curve (AUC_0-48h) for D, DA and DOH was estimated as 37, 335 and 27,579 h∙nmol/L, respectively. Gender-wise, men absorbed the parent compounds faster and took shorter time to reach DOH peak concentration. The human data supported an extensive conversion of D and DA to DOH, even though they metabolized DA slightly slower than rodents. Therefore, the data generated in rodent models concerning anti-cancer efficacy, safety, tissue distribution and pharmacodynamic biomarkers will likely be relevant for human translation.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT02114957","term_id":"NCT02114957"}}NCT02114957     

Statistical Analysis of Crystallization Database Links Protein Physico-Chemical Features with Crystallization Mechanisms

X-ray crystallography is the predominant method for obtaining atomic-scale information about biological macromolecules. Despite the success of the technique, obtaining well diffracting crystals still critically limits going from protein to structure. In practice, the crystallization process proceeds through knowledge-informed empiricism. Better physico-chemical understanding remains elusive because of the large number of variables involved, hence little guidance is available to systematically identify solution conditions that promote crystallization. To help determine relationships between macromolecular properties and their crystallization propensity, we have trained statistical models on samples for 182 proteins supplied by the Northeast Structural Genomics consortium. Gaussian processes, which capture trends beyond the reach of linear statistical models, distinguish between two main physico-chemical mechanisms driving crystallization. One is characterized by low levels of side chain entropy and has been extensively reported in the literature. The other identifies specific electrostatic interactions not previously described in the crystallization context. Because evidence for two distinct mechanisms can be gleaned both from crystal contacts and from solution conditions leading to successful crystallization, the model offers future avenues for optimizing crystallization screens based on partial structural information. The availability of crystallization data coupled with structural outcomes analyzed through state-of-the-art statistical models may thus guide macromolecular crystallization toward a more rational basis.     

Reductive dechlorination of chlorophenols in slurries of low-organic-carbon marine sediments and subsurface soils

The reductive dechlorination of 2,4- and 3,4-dichlorophenol (DCP) was studied in slurries of marine sediments and subsurface soils with dissolved organic carbon concentrations less than 1 ppm. Dechlorination was markedly greater in marine sediment slurries than in subsoil slurries, although similar products were observed in each case. From 25% to 98% of the 2,4- and 3,4-DCP (6.5 μm/l) added to most marine slurries was converted to 4- and 3-chlorophenol (CP) respectively, within 30 weeks. In contrast 2,4-DCP was dechlorinated to 4-CP (>90%) in only 1 of 24 replicate subsoil slurries after 32 weeks of incubation. Dechlorination was observed within 2 weeks when yeast extract was added to subsoil slurries; yeast extract additions also stimulated dechlorination in marine sediments but to a lesser extent. The intermediate monochlorophenol products did not persist in marine slurries but did persist in the subsoil slurries. It was concluded that the total organic carbon at a site is not always a good predictor of the site's ability to support dechlorination activity. Received: 3 December 1996 / Received revision: 28 February 1997 / Accepted: 7 March 1997     

Receptor-type Protein-tyrosine Phosphatase ζ Is a Functional Receptor for Interleukin-34

Interleukin-34 (IL-34) is highly expressed in brain. IL-34 signaling via its cognate receptor, colony-stimulating factor-1 receptor (CSF-1R), is required for the development of microglia. However, the differential expression of IL-34 and the CSF-1R in brain suggests that IL-34 may signal via an alternate receptor. By IL-34 affinity chromatography of solubilized mouse brain membrane followed by mass spectrometric analysis, we identified receptor-type protein-tyrosine phosphatase ζ (PTP-ζ), a cell surface chondroitin sulfate (CS) proteoglycan, as a novel IL-34 receptor. PTP-ζ is primarily expressed on neural progenitors and glial cells and is highly expressed in human glioblastomas. IL-34 selectively bound PTP-ζ in CSF-1R-deficient U251 human glioblastoma cell lysates and inhibited the proliferation, clonogenicity, and motility of U251 cells in a PTP-ζ-dependent manner. These effects were correlated with an increase in tyrosine phosphorylation of the previously identified PTP-ζ downstream effectors focal adhesion kinase and paxillin. IL-34 binding to U251 cells was abrogated by chondroitinase ABC treatment, and CS competed with IL-34 for binding to the extracellular domain of PTP-ζ and to the cells, indicating a dependence of binding on PTP-ζ CS moieties. This study identifies an alternate receptor for IL-34 that may mediate its action on novel cellular targets.     

Recombinant BCG Expressing Mycobacterium ulcerans Ag85A Imparts Enhanced Protection against Experimental Buruli ulcer

Buruli ulcer, an emerging tropical disease caused by Mycobacterium ulcerans (MU), is characterized by disfiguring skin necrosis and high morbidity. Relatively little is understood about the mode of transmission, pathogenesis, or host immune responses to MU infection. Due to significant reduction in quality of life for patients with extensive tissue scarring, and that a disproportionately high percentage of those affected are disadvantaged children, a Buruli ulcer vaccine would be greatly beneficial to the worldwide community. Previous studies have shown that mice inoculated with either M. bovis bacille Calmette–Guérin (BCG) or a DNA vaccine encoding the M. ulcerans mycolyl transferase, Ag85A (MU-Ag85A), are transiently protected against pathology caused by intradermal challenge with MU. Building upon this principle, we have generated quality-controlled, live-recombinant strains of BCG and M. smegmatis which express the immunodominant MU Ag85A. Priming with rBCG MU-Ag85A followed by an M. smegmatis MU-Ag85A boost strongly induced murine antigen-specific CD4⁺ T cells and elicited functional IFNγ-producing splenocytes which recognized MU-Ag85A peptide and whole M. ulcerans better than a BCG prime-boost vaccination. Strikingly, mice vaccinated with a single subcutaneous dose of BCG MU-Ag85A or prime-boost displayed significantly enhanced survival, reduced tissue pathology, and lower bacterial load compared to mice vaccinated with BCG. Importantly, this level of superior protection against experimental Buruli ulcer compared to BCG has not previously been achieved. These results suggest that use of BCG as a recombinant vehicle expressing MU antigens represents an effective Buruli ulcer vaccine strategy and warrants further antigen discovery to improve vaccine efficacy.     

Labor and the Human Relationship with Nature: The Naturalization of Politics in the Work of Thomas Henry Huxley, Herbert George Wells, and William Morris

Historically labor has been central to humaninteractions with the environment, yetenvironmentalists pay it scant attention. Indeed, they have been critical of those whoforeground labor in their politics, socialistsin particular. However, environmentalists havefound the nineteenth-century socialist WilliamMorris appealing despite the fact that he wroteextensively on labor. This paper considers theplace of labor in the relationship betweenhumanity and the natural world in the work ofMorris and two of his contemporaries, theeminent scientist Thomas Henry Huxley, and theFabian socialist Herbert George Wells. Isuggest that Morris's conception of labor hasmuch to recommend it to environmentalists whoare also interested in issues of socialjustice.     

Individual muscle force parameters and fiber operating ranges for elbow flexion-extension and forearm pronation-supination

We have quantified individual muscle force and moment contributions to net joint moments and estimated the operating ranges of the individual muscle fibers over the full range of motion for elbow flexion/extension and forearm pronation/supination. A three dimensional computer graphics model was developed in order to estimate individual muscle contributions in each degree of freedom over the full range of motion generated by 17 muscles crossing the elbow and forearm. Optimal fiber length, tendon slack length, and muscle specific tension values were adjusted within the literature range from cadaver studies such that the net isometric joint moments of the model approximated experimental joint moments within one standard deviation. Analysis of the model revealed that the muscles operate on varying portions of the ascending limb, plateau region, and descending limb of the force-length curve. This model can be used to further understand isometric force and moment contributions of individual muscles to net joint moments of the arm and forearm and can serve as a comprehensive reference for the forces and moments generated by 17 major muscles crossing the elbow and wrist.     

Leukemia inhibitory factor, oncostatin M, IL-6, and stem cell factor mRNA expression in human thymus increases with age and is associated with thymic atrophy

The roles that thymus cytokines might play in regulating thymic atrophy are not known. Reversing thymic atrophy is important for immune reconstitution in adults. We have studied cytokine mRNA steady-state levels in 45 normal human (aged 3 days to 78 years) and 34 myasthenia gravis thymuses (aged 4 to 75 years) during aging, and correlated cytokine mRNA levels with thymic signal joint (sj) TCR delta excision circle (TREC) levels, a molecular marker for active thymopoiesis. LIF, oncostatin M (OSM), IL-6, M-CSF, and stem cell factor (SCF) mRNA were elevated in normal and myasthenia gravis-aged thymuses, and correlated with decreased levels of thymopoiesis, as determined by either decreased keratin-positive thymic epithelial space or decreased thymic sjTRECs. IL-7 is a key cytokine required during the early stages of thymocyte development. Interestingly, IL-7 mRNA expression did not fall with aging in either normal or myasthenia gravis thymuses. In vivo administration of LIF, OSM, IL-6, or SCF, but not M-CSF, i.p. to mice over 3 days induced thymic atrophy with loss of CD4+, CD8+ cortical thymocytes. Taken together, these data suggest a role for thymic cytokines in the process of thymic atrophy.     

Contrasting patterns of genetic structure and evolutionary history as revealed by rnitochondrial DNA and nuclear gene-enzyme variation betweenDrosophila melanogaster andDrosophila simulans

Drosophila melanogaster and its sibling speciesD. simulans have a cosmopolitan distribution. Studies on nuclear gene-enzyme variation from natural populations of these species reveal that the two have almost equal overall heterozygosity, yetD. simulans populations are significantly less differentiated. However, it is not clear whether this difference in population structure represents a difference in the genetic strategy with which they respond to the same adaptive challenges, or is the result of difference in species history. To help answer this question, we have undertaken an intensive survey of restriction fragment length polymorphisms of mitochondrial DNA (mtDNA) fromD. simulans; the results are compared with those fromD. melanogaster. We surveyed 69 isofemale lines ofD. simulans from four continents and seven lines from the Seychelles Islands. Ten restriction enzymes detected 104 restriction sites in the continental mtDNAs, of which only threeHinf1 sites were variable and account for fourHlnf1 (restriction variants) haplotypes. These four variants were all found in geographically distant locations. By contrast, twenty-three haplotypes were observed inD. melanogaster, many of which were observed in only one population. It would seem, therefore, that these two species have had different histories. Specifically, cosmopolitan populations ofD: simulans are probably products of a comparatively recent expansion from a source population in Africa. These results do not negate differences in their genetic strategy of adaptation, but they do show the importance of historical contingency in the present-day pattern of geographic variation.