Biomedical subjects

S-X Lin

Publications and source records attributed to S-X Lin.

8 recordsLinked to original sources

PubMed2006

Crystallization and preliminary X-ray diffraction analysis of E. coli arginyl-tRNA synthetase in complex form with a tRNAArg.

Amino acids are building blocks of proteins, while aminoacyl-tRNA synthetases (aaRSs) catalyze the first reaction in such building: the biosynthesis of proteins. The E. coli arginyl-tRNA synthetase (ArgRS) has been crystallized in complex form with tRNA(Arg) (B. stearothermophilus), at pH 5.6 using ammonium sulfate as a precipitating agent. Two crystal forms have been identified based on unit cell dimension. The complete data sets from both crystal forms have been collected with a primitive hexagonal space group. A data set of Form II crystals at 3.2 A and 94% completeness has been obtained, with unit cell parameters a = b = 98.0 A, c = 463.2 A, and alpha = beta = 90 degrees , gamma = 120 degrees , being different from a = b = 110.8 A, c = 377.8 A for form I. The structure determination will demonstrate the interaction of these two macromolecules to understand the special mechanism of ArgRS that requires the presence of tRNA for amino acid activation. Such complex structure also provides a wide opening for inhibitor search using bioinformatics.

Arginine-tRNA Ligase↗

PubMed2006

Attempts to rationalize protein crystallization using relative crystallizability.

Protein crystal growth (PCG) remains the bottleneck of crystallography despite many decades of study. The nucleation zone in the two-dimensional-phase diagram has been used to evaluate the relative crystallizability of proteins, which is expressed as a percentage over the phase area delineated by experimental protein and precipitating agent concentration ranges. For protein-salts which are subject to a direct temperature effect on solubility, as represented by Egg Lysozyme, a decrease in temperature augments the nucleation zone percentage whereas for those with retrograde solubility as a function of temperature, for example fructose-1,6-bisphosphatase in the presence and absence of AMP, an increase in temperature can significantly enhance the relative crystallizability. These results have been confirmed by the number of "hits" using PEGs as precipitating agents in Sparse Matrix Screen experiments for different proteins and are in excellent agreement with the relative crystallizability. The relationship between solubility dependence, relative crystallizability and crystallization success, has been evidenced. Such crystallizability can become a guide to identify efficient crystallization regions, providing a rational approach to PCG and structural biology.

Adenosine Monophosphate↗

PubMed2006

Structural basis of the multispecificity demonstrated by 17beta-hydroxysteroid dehydrogenase types 1 and 5.

17Beta-hydroxysteroid dehydrogenases/ketosteroid reductases (17beta-HSDs/KSRs) catalyze the last step of sex steroid synthesis or the first step of their degradation, and are thus critical for many physiological processes. The multispecificity demonstrated by 17beta-HSDs is important for steroid metabolism in gonadal and peripheral tissues, and is a consequence of the architecture of their binding and catalytic sites. Structurally, most of the family members are short chain dehydrogenase-reductases (SDRs) except the type 5 enzyme, which is an aldo-keto reductase (AKR). 17Beta-HSD type 1, a representative of the SDR family, has been studied extensively since the 1950s. However, its structure was not determined until the 1990s. It has always been considered as estrogen specific, in accord with the narrow binding tunnel that has been structurally determined and has been found to be complementary to estrogens. A recent study revealed that, in spite of the enzyme's narrow binding tunnel, the pseudo-symmetry of C19 steroids leads to its alternative binding, resulting in the multispecificity of the enzyme. Expressed in ovary, breast and placenta, the enzyme catalyzes the formation of another estrogen A-diol from DHEA in addition to the biosynthesis of estradiol; it also inactivates the most active androgen DHT by both 17beta-hydroxysteroid oxidation and 3-ketosteroid reduction. Type 5 17beta-HSD (AKR1C3) differs significantly from the type 1 enzyme by possessing a spacious and flexible steroid-binding site. This is estimated to be about 960 or 470 A3 in ternary complex with testosterone or 4-dione, respectively, whereas the binding site volume of 17beta-HSD1 is only about 340 A3. This characteristic of the 17beta-HSD5 binding site permits the docking of various steroids in different orientations, which encompasses a wider range of activities from 20alpha-, 17beta- and 3alpha-HSD/KSR to prostaglandin 11-ketoreductase. The in vitro activities of the enzyme are significantly lower than the type 1 enzyme. In the ternary complex with testosterone, the steroid C3-C17 position is quasi-reversed as compared to the complex with 4-dione. The multi-specificity contributes significantly to steroid metabolism in peripheral tissues, due to the high levels of 17beta-HSD5 mRNA in both breast and prostate tissues.

17-Hydroxysteroid Dehydrogenases↗

PubMed2006

Autoimmunity to type VII collagen in SKH1 mice is independent of regulatory T cells.

Epidermolysis bullosa acquisita is an autoimmune blistering disease characterized by circulating and skin basement membrane-bound IgG autoantibodies to type VII collagen, a major structural protein of the dermal-epidermal junction. Regulatory T cells (T(reg)) suppress self antigen-mediated autoimmune responses. To investigate the role of T(reg) in the the autoimmune response to type VII collagen in a mouse model, a monoclonal antibody against mouse CD25 was used to deplete T(reg). A recombinant mouse type VII collagen NC1 domain protein and mouse albumin were used as antigens. SKH1 mice were used as a testing host. Group 1 mice received NC1 immunization and were functionally depleted of T(reg); group 2 mice received NC1 immunization and rat isotype control; and group 3 mice received albumin immunization and were functionally depleted of T(reg). Results demonstrated that anti-NC1 IgG autoantibodies with high titres, as determined by enzyme-linked immunosorbent assay and Western blotting, developed in all mice immunized with NC1 (groups 1 and 2), but were undetected in group 3 mice. The predominant subclasses of anti-NC1 autoantibodies were IgG1, IgG2a and IgG2b; furthermore, these antibodies carried only the kappa light chain. IgG autoantibodies in the sera of NC1-immunized mice reacted with mouse skin basement membrane in vitro and deposited in skin basement membrane in vivo as detected by indirect and direct immunofluorescence microscopy, respectively. Our data suggest that the development of autoimmunity against type VII collagen in mice is independent of T(reg) function and the autoimmune response is mediated by both Th1 and Th2 cells. We speculate that the basement membrane deposition of IgG may eventually lead to blister development.

Animals↗

PubMed2005

Correlation of disease evolution with progressive inflammatory cell activation and migration in the IL-4 transgenic mouse model of atopic dermatitis.

Atopic dermatitis is a chronic inflammatory skin disease characterized by inflammatory cell infiltration in the skin. In order to assess the roles of inflammatory cells in this disease, we analysed the activation status and surface markers of various leucocytes in the IL-4 transgenic mouse model of atopic dermatitis, by flow cytometry, immuofluorescence microscopy, and T cell proliferation assays. The studies were performed with a nontransgenic mouse control and transgenic mice at three disease stages: before disease onset, early skin disease, and late skin disease, so that we can delineate the immunological sequence of events. As the skin disease evolves, the skin draining lymph node cells from IL-4-Tg mice show a spontaneous proliferation and a progressively enhanced proliferative response to stimulants including anti-CD3, Con A, PHA, and Staphylococcus enterotoxins A and B. As the disease evolves, the percent of lymphoid organ T cells expressing activation molecules (CD44 and CD69) and costimulatory molecules (ICOS and PD-1) are progressively increased; the percent and total number of T cells are reduced in an incremental manner in the secondary lymphoid organs while the number of T cells infiltrating the skin increases in an incremental fashion; the total number of dendritic antigen presenting cells, macrophages, and NK cells gradually increases in the lymphoid organs. Collectively, our results suggest that there is a continued and progressive migration of activated inflammatory cells from the secondary lymphoid organs into the skin where they participate in immune responses resulting in the pathology associated with inflammation.

Animals↗

PubMed2005

The disease progression in the keratin 14 IL-4-transgenic mouse model of atopic dermatitis parallels the up-regulation of B cell activation molecules, proliferation and surface and serum IgE.

We have previously characterized the keratin 14 interleukin-4-transgenic (IL-4-Tg) mouse model of atopic dermatitis as a chronic pruritic inflammatory skin disease typified by skin infiltration of inflammatory cells and early up-regulation of Th2 cytokines and late surge of Th1 cytokines. In the present study, we examined the involvement of B cells. Systematic examinations of the following immunological parameters on B cells were carried out in non-Tg control mice and in IL-4-Tg mice at before disease onset and early and late disease stages so that we could determine the immunological sequence of events leading to the disease development: surface expressions of IA/IE, activation and costimulatory molecules, proliferation under LPS or IgM stimulation, quantification of cell surface and serum IgE, IgG1, and IgG2a. Our results showed that as the disease progresses from before onset to early disease and to late disease, there is a parallel increase in surface markers of B cell activation (IA/IE, CD44, CD69, CD80 and CD86), in B cell proliferation, and in cell surface and serum IgE. Significant increases of Th2-driven serum IgG1 and IgE in early disease was followed by significant increase of Th1-driven IgG2a in late disease. Importantly the significant increases of activation molecule (IA/IE), proliferation (to LPS), and surface IgE on B cells of the IL-4-Tg mice precedes the up-regulation of serum IgE and disease onset. These data suggest that activated B cells may play a role in atopic dermatitis disease development by up-regulating serum IgE concentration, which serves as a marker of disease onset.

Animals↗

PubMed2005

Is dehydroepiandrosterone a hormone?

Dehydroepiandrosterone (DHEA) is not a hormone but it is a very important prohormone secreted in large amounts by the adrenals in humans and other primates, but not in lower species. It is secreted in larger quantities than cortisol and is present in the blood at concentrations only second to cholesterol. All the enzymes required to transform DHEA into androgens and/or estrogens are expressed in a cell-specific manner in a large series of peripheral target tissues, thus permitting all androgen-sensitive and estrogen-sensitive tissues to make locally and control the intracellular levels of sex steroids according to local needs. This new field of endocrinology has been called intracrinology. In women, after menopause, all estrogens and almost all androgens are made locally in peripheral tissues from DHEA which indirectly exerts effects, among others, on bone formation, adiposity, muscle, insulin and glucose metabolism, skin, libido and well-being. In men, where the secretion of androgens by the testicles continues for life, the contribution of DHEA to androgens has been best evaluated in the prostate where about 50% of androgens are made locally from DHEA. Such knowledge has led to the development of combined androgen blockade (CAB), a treatment which adds a pure anti-androgen to medical (GnRH agonist) or surgical castration in order to block the access of the androgens made locally to the androgen receptor. In fact, CAB has been the first treatment demonstrated to prolong life in advanced prostate cancer while recent data indicate that it can permit long-term control and probably cure in at least 90% of cases of localized prostate cancer. The new field of intracrinology or local formation of sex steroids from DHEA in target tissues has permitted major advances in the treatment of the two most frequent cancers, namely breast and prostate cancer, while its potential use as a physiological HRT could well provide a physiological balance of androgens and estrogens, thus offering exciting possibilities for women's health at menopause.

Adult↗

PubMed2004

Protein crystal growth on board Shenzhou 3: a concerted effort improves crystal diffraction quality and facilitates structure determination.

The crystallization of 16 proteins was carried out using 60 wells on board Shenzhou 3 in 2002. Although the mission was only 7 days, careful and concerted planning at all stages made it possible to obtain crystals of improved quality compared to their ground controls for some of the proteins. Significantly improved resolutions were obtained from diffracted crystals of 4 proteins. A complete data set from a space crystal of the PEP carboxykinase yielded significantly higher resolution (1.46A vs. 1.87A), I/sigma (22.4 vs. 15.5), and a lower average temperature factor (29.2A(2) vs. 42.9A(2)) than the best ground-based control crystal. The 3-D structure of the enzyme is well improved with significant ligand density. It has been postulated that the reduced convection and absence of macromolecule sedimentation under microgravity have advantages/benefits for protein crystal growth. Improvements in experimental design for protein crystal growth in microgravity are ongoing.

Crystallization↗