Determination of free and amidated bile acids by high-performance liquid chromatography with evaporative light-scattering mass detection.

A simple reverse phase high-performance liquid chromatographic method for a simultaneous analysis of free, glycine- and taurine-amidated bile acids is described. The resolution of ursodeoxycholic, cholic, chenodeocycholic, deoxycholic, and lithocholic acids, either free or amidated with glycine and taurine, is achieved using a C-18 octadecylsilane column (30 cm length, 4 micron particle size) with a gradient elution of aqueous methanol (65----75%) containing 15 mM ammonium acetate, pH 5.40, at 37 degrees C. The separated bile acids are detected with a new evaporative light-scattering mass detector and by absorbance at 200 nm. A complete resolution of the 16 bile acids, including the internal standard nor-deoxycholic acid, is obtained within 55 min. Using the light-scattering mass detector, amidated bile acids and, for the first time, free bile acids can be detected with similar detection limits in the order of 2-7 nmol. The new detector improves the baseline and the signal-to-noise ratio over the UV detection as it is not affected by impurities present in the samples with higher molar absorptivity than bile acids or by the change in the mobile phase composition during the gradient. The method fulfills all the standard requirements of precision and accuracy and the linearity of the mass detector is over 5 decade the detection limit. The new method has been used for the direct analysis of bile acid in stools and bile with only a preliminary clean-up procedure using a C-18 reverse phase extraction.     

In vitro membrane association of the F0 polypeptides of the Escherichia coli proton translocating ATPase.

The F0 polypeptides a, b, and c of the H+-translocating ATPase associated with membranes when synthesized in vitro. This association occurred when the membranes were present either cotranslationally or post-translationally. In addition, the F0 polypeptides associated with liposomes. The membrane association seemed to be an insertion process since there was protection of polypeptides a and c from proteolysis. The in vitro insertion of the F0 polypeptides a, b, and c was independent of the synthesis of each polypeptide and of the F1 polypeptides.     

Gene expression profiling of mouse Sertoli cell lines

The proliferation and differentiation of Sertoli cells is regulated by follicle-stimulating hormone (FSH). The molecular events following FSH stimulation are only partially known. To investigate FSH action in Sertoli cells, we established two novel FSH-responsive mouse Sertoli-cell-derived lines expressing human wild-type (WT) FSH receptor (FSHR) or overexpressing mutated (Asp567Gly) constitutively active FSHR (MUT). Gene expression profiling with commercially available cDNA arrays, including 588 mouse genes, revealed 146 genes expressed in both cell lines. Compared with the expression pattern of WT cells, 20 genes were identified as being either up- or down-regulated (>two-fold) in the MUT cells. We observed a strong differential expression of factors involved in cellular proliferation, e.g. cyclin D2 (repressed to nearly undetectable levels), proliferating cell nuclear antigen (2.5-fold repression) and Eps-8 (six-fold repression), and in genes involved in cellular differentiation, e.g. cytokeratin-18 (13-fold induction). The cDNA array results for six representative genes were confirmed by Northern blotting, which also included the parental SK-11 cell line devoid of FSHR expression. We found no further acute FSH- or forskolin-induced change in expression levels after 3-h stimulations, suggesting that the observed differences between the two cell lines is a consequence of mild, chronically increased, cAMP production in MUT cells. These results provide a platform for the further investigation of selected candidate genes in primary cultures and/or in vivo.Electronic Supplementary Material Supplementary material is available in the online version of this article at This work was supported by grants from the Deutsche Forschungsgemeinschaft (Confocal Research Group The Male Gamete: Production, Maturation, Function, grant FOR 197-3) and from the German Academic Exchange Service (DAAD) to P. Mathur     

Pharmacokinetics and pharmacodynamics of injectable testosterone undecanoate in castrated cynomolgus monkeys (Macaca fascicularis) are independent of different oil vehicles

Testosterone undecanoate (TU) dissolved in soybean oil was developed in China to improve the pharmacokinetics of this testosterone ester in comparison with TU in castor or tea seed oil. As a pre-clinical primate model, three groups of five castrated cynomolgus macaques received either a single intramuscular injection of 10 mg/kg body weight TU in soybean oil, in tea seed oil, or in castor oil (equals 6.3 mg pure T/kg body weight for all preparations). Testosterone, estradiol, luteinizing hormone, and follicle-stimulating hormone as well as prostate volume, body weight and ejaculate weight were evaluated. After injection supraphysiological testosterone levels were induced. There were no significant differences in the pharmacokinetics of the three TU preparations for testosterone and estradiol. The gonadotropin levels showed a high individual variation. Prostate volumes increased equally in all groups after administration and declined to castrate level afterwards. The results suggest that TU in soybean oil produces similar effects as TU in the other vehicles. This study in non-human primates provides no objection to testing of this new preparation in humans.     

The beta subunit of the Escherichia coli ATP synthase exhibits a tight membrane binding property

We have developed a chromatographic procedure to analyze the association of the subunits of the Escherichia coli F1Fo-ATP synthase with the cytoplasmic membrane. Minicells containing [35S]-labeled ATP synthase subunits are treated with lysozyme, solubilized, and chromatographed on a Sepharose CL-2B column in buffer containing urea and taurodeoxycholate. ATP synthase subunits are resolved into membrane intrinsic and membrane extrinsic subunits. Interestingly, a significant amount (36%) of the F1 subunit beta fractionates with the membrane intrinsic Fo subunits. About half of this amount (19%) of beta is non-specifically bound to the membrane. Interaction of beta with the membrane is not mediated by the amino terminal portion of beta.     

Thioredoxin Is Involved in Endothelial Cell Extracellular Transglutaminase 2 Activation Mediated by Celiac Disease Patient IgA

Purpose

To investigate the role of thioredoxin (TRX), a novel regulator of extracellular transglutaminase 2 (TG2), in celiac patients IgA (CD IgA) mediated TG2 enzymatic activation.

Methods

TG2 enzymatic activity was evaluated in endothelial cells (HUVECs) under different experimental conditions by ELISA and Western blotting. Extracellular TG2 expression was studied by ELISA and immunofluorescence. TRX was analysed by Western blotting and ELISA. Serum immunoglobulins class A from healthy subjects (H IgA) were used as controls. Extracellular TG2 enzymatic activity was inhibited by R281. PX12, a TRX inhibitor, was also employed in the present study.

Results

We have found that in HUVECs CD IgA is able to induce the activation of extracellular TG2 in a dose-dependent manner. Particularly, we noted that the extracellular modulation of TG2 activity mediated by CD IgA occurred only under reducing conditions, also needed to maintain antibody binding. Furthermore, CD IgA-treated HUVECs were characterized by a slightly augmented TG2 surface expression which was independent from extracellular TG2 activation. We also observed that HUVECs cultured in the presence of CD IgA evinced decreased TRX surface expression, coupled with increased secretion of the protein into the culture medium. Intriguingly, inhibition of TRX after CD IgA treatment was able to overcome most of the CD IgA-mediated effects including the TG2 extracellular transamidase activity.

Conclusions

Altogether our findings suggest that in endothelial cells CD IgA mediate the constitutive activation of extracellular TG2 by a mechanism involving the redox sensor protein TRX.     

Enhancing our Understanding of Protein Structure: the Work of Jane and David Richardson

Jane and David Richardson have worked together since 1964, and their research has made strides in enhancing our understanding of protein structure. Two of the crystal structures they solved are staphylococcal nuclease and copper, zinc superoxide dismutase, both of which were published in the Journal of Biological Chemistry Classic papers reprinted here.A High Resolution Structure of an Inhibitor Complex of the Extracellular Nuclease of Staphylococcus aureus. I. Experimental Procedures and Chain Tracing (Arnone, A., Bier, C. J., Cotton, F. A., Day, V. W., Hazen, E. E., Jr., Richardson, D. C., Richardson, J. S., and, in part, Yonath, A. (1971) J. Biol. Chem. 246, 2302–2316)The Crystal Structure of Bovine Cu²⁺,Zn²⁺ Superoxide Dismutase at 5.5-Å Resolution (Thomas, K. A., Rubin, B. H., Bier, C. J., Richardson, J. S., and Richardson, D. C. (1974) J. Biol. Chem. 249, 5677–5683)From an early age, Jane Shelby Richardson was interested in astronomy; she came in third in the Westinghouse Science Talent Search by calculating the orbit of the Sputnik satellite from her own observations. Wanting to continue her science education, she enrolled at Swarthmore College as a math, physics, and astronomy major. However, she became interested in philosophy and ended up switching majors. It was at Swarthmore that she met David Richardson. Both Jane and David graduated in 1962, and they married a year later. David went on to the Massachusetts Institute of Technology to work on a doctorate in chemistry while Jane went to Harvard University to get a degree in philosophy. Opting out after a year with a Master''s degree, Jane tried teaching high school science and in 1964 settled in to a job as a technician in the lab where David was doing graduate research.Open in a separate windowJane and David RichardsonWhen David was accepted at MIT, he decided to join the laboratory of F. Albert Cotton, who was working on small molecule inorganic chemistry and crystallography. Having become intrigued by biochemistry, David wanted to solve the structure of a protein and settled on staphylococcal nuclease upon the suggestion of Nobel laureate and Journal of Biological Chemistry (JBC) Classic author Christian Anfinsen (1). At that point, only two protein structures had been solved, those of hemoglobin and myoglobin. The methodology of protein crystallography was still in its infancy, so David and Jane along with Ted Hazen tested crystal growth conditions one at a time in batch (2), modified a small molecule x-ray diffractometer (controlled by punched paper tape) to take good protein data, wrote all their own software, and first learned what amino acids look like in 2 Å electron density when their structure was solved.After working for 7 years, they determined the structure of the nuclease in 1969, as reported in the first JBC Classic reprinted here, tying with Eisenberg and Dickerson''s cytochrome c for the 10th new protein structure. The 2 Å resolution staphylococcal nuclease structure showed the arrangement of β and helical features, the presence of disordered terminal tails and a partially disordered loop, and the geometry of binding a Ca²⁺ and the thymidine 3′,5′-diphosphate inhibitor at the active site. As envisioned by Anfinsen, this relatively simple single-domain protein did indeed become an important model system for the study of protein folding, first at NIH and later at Johns Hopkins.Shortly after this feat, David was offered a faculty position in biochemistry at Duke University. He and Jane established a lab there and started working on the structure of copper,zinc superoxide dismutase, which was a favorite enzyme at Duke (its function was discovered by JBC Classic author Irwin Fridovich (3), the sequence was solved in JBC Classic author Robert L. Hill''s lab (4), and a number of other people in the department have worked on it). Superoxide dismutases catalyze the dismutation of superoxide into oxygen and hydrogen peroxide, making them an important antioxidant defense in nearly all cells exposed to oxygen.By 1972, David and Jane were able to get crystals of the bovine superoxide dismutase with strong diffraction out to 2 Å, published in a short communication in the JBC (5). Two years later, they solved the structure of the enzyme at 5.5 Å, as reported in the second JBC Classic reprinted here. From their data, they concluded that each subunit of the dimeric enzyme had extensive β structure surrounding a central hydrophobic core, and they were able to determine a probable location for the zinc ion from a mercury for zinc substitution. They traced the polypeptide chain from further data at 3 Å, showing that the protein''s dominant structural feature was an 8-stranded barrel of antiparallel β-pleated sheet and that the copper and zinc were only 6 Å apart at the active site, sharing a His ligand. The β-sheet topology was compared with immunoglobulins, nuclease, and others, leading to definition of the “Greek key” β-barrel fold (6). At 2 Å resolution, the overall structure and active site were described in detail, including electrostatic guidance of substrate (7).In the 1980s the Richardsons started exploring synthetic biochemistry and computational biology and helped open up the field of protein de novo design. In the 1990s they pioneered molecular graphics for personal computers by developing the kinemage system of molecular graphics and the Mage program to display them on small computers, and they developed all-atom contact analysis to measure goodness of fit inside proteins and in interactions with surrounding molecules. The Richardson lab currently studies structural motifs in RNA as well as proteins, as part of the RNA Ontology Consortium, collaborates in development of the Phenix crystallographic software system, and hosts the MolProbity web service for validation and accuracy improvement of protein and RNA experimental structures.In addition to the accomplishments above, Jane is widely known for her creation of ribbon drawings to schematize protein 3D structures, first published in Advances in Protein Chemistry in 1981 (8). The drawings stemmed from Jane''s realization that a general classification scheme could be developed from the recurring patterns of structural motifs within the “folds” of proteins. She created ribbon drawings of those folds, making a uniform set of conventions for drawing the 75 protein structures that had been solved at that time, including the superoxide dismutase shown in Fig. 1. Computer versions of the ribbon representation have since become one of the most common ways of visualizing proteins.Open in a separate windowFIGURE 1.Hand-drawn ribbon schematic of the Cu,Zn-superoxide dismutase subunit, by Jane Richardson.Jane and David remain in the Department of Biochemistry at Duke University, where Jane is a James B. Duke professor and David is a professor. Although Jane does not have a Ph.D. degree, she has been given three honorary doctorates, from Swarthmore College, the University of North Carolina Chapel Hill, and the University of Richmond. In 1985 she was awarded a MacArthur Fellowship for her work in structural biology, and she was elected to the National Academy of Sciences and the American Academy of Arts and Sciences in 1991 and to the Institute of Medicine in 2006. In 2010, Jane was elected president of the Biophysical Society. She also is the co-recipient of the Protein Society''s Amgen Award with David (1995) and the Biophysical Society''s Emily M. Gray Award (2001). David is the founding director of the Structural Biology and Biophysics Graduate Training Program at Duke and also has received numerous honors including Science Digest''s 100 Best Innovations of 1985, a BioTechnology Winter Symposium Special Achievement Award (1995), and the Duke University Gordon Hammes Teaching and Mentoring Award (2009).