Biomedical subjects

Y Xue

Publications and source records attributed to Y Xue.

At least 73 records · Page 4Linked to original sources

PubMed2000

A strategically positioned cation is crucial for efficient catalysis by chorismate mutase.

Combinatorial mutagenesis and in vivo selection experiments previously afforded functional variants of the AroH class Bacillus subtilis chorismate mutase lacking the otherwise highly conserved active site residue Arg(90). Here, we present a detailed kinetic and crystallographic study of several such variants. Removing the arginine side chain (R90G and R90A) reduced catalytic efficiency by more than 5 orders of magnitude. Reintroducing a positive charge to the active site through lysine substitutions restored more than a factor of a thousand in k(cat). Remarkably, the lysine could be placed at position 90 or at the more remote position 88 provided a sterically suitable residue was present at the partner site. Crystal structures of the double mutants C88S/R90K and C88K/R90S show that the lysine adopts an extended conformation that would place its epsilon-ammonium group within hydrogen-bonding distance of the ether oxygen of bound chorismate in the transition state. These results provide support for the hypothesis that developing negative charge in the highly polarized transition state is stabilized electrostatically by a strategically placed cation. The implications of this finding for the mechanism of all natural chorismate mutases and for the design of artificial catalysts are discussed.

Binding Sites↗

PubMed2000

Purification and initial characterization of RNA polymerase from Thermus thermophilus strain HB8.

Utilizing a novel and rapid two-column purification procedure, the DNA-dependent RNA polymerase (RNAP) from the thermophile, Thermus thermophilus HB8, was purified to electrophoretic homogeneity with a recovery of 65% (as determined by RNAP activity) in less than 2 days. The purified enzyme was characterized using DNA containing the lambdaP(R) promoter. KMnO(4) footprinting, abortive initiation assays, and the formation of the specific stalled elongation complex provide compelling evidence that T. thermophilus RNA polymerase can bind to DNA containing the lambdaP(R) promoter, form an open complex, and initiate transcription in a temperature-dependent manner. This evidence suggests that T. thermophilus RNAP possesses less intrinsic binding energy than E. coli RNAP. Instead, T. thermophilus relies on the high temperatures of its environment to provide the thermal energy required to stimulate open promoter complex formation, initiate transcription, and facilitate the conformational changes in RNA polymerase that result in nucleotide incorporation.

Bacteriophage lambda↗

PubMed2000

The human SWI/SNF-B chromatin-remodeling complex is related to yeast rsc and localizes at kinetochores of mitotic chromosomes.

The SWI/SNF family of chromatin-remodeling complexes facilitates gene expression by helping transcription factors gain access to their targets in chromatin. SWI/SNF and Rsc are distinctive members of this family from yeast. They have similar protein components and catalytic activities but differ in biological function. Rsc is required for cell cycle progression through mitosis, whereas SWI/SNF is not. Human complexes of this family have also been identified, which have often been considered related to yeast SWI/SNF. However, all human subunits identified to date are equally similar to components of both SWI/SNF and Rsc, leaving open the possibility that some or all of the human complexes are rather related to Rsc. Here, we present evidence that the previously identified human SWI/SNF-B complex is indeed of the Rsc type. It contains six components conserved in both Rsc and SWI/SNF. Importantly, it has a unique subunit, BAF180, that harbors a distinctive set of structural motifs characteristic of three components of Rsc. Of the two mammalian ATPases known to be related to those in the yeast complexes, human SWI/SNF-B contains only the homolog that functions like Rsc during cell growth. Immunofluorescence studies with a BAF180 antibody revealed that SWI/SNF-B localizes at the kinetochores of chromosomes during mitosis. Our data suggest that SWI/SNF-B and Rsc represent a novel subfamily of chromatin-remodeling complexes conserved from yeast to human, and could participate in cell division at kinetochores of mitotic chromosomes.

Adenosine Triphosphatases↗

PubMed2000

Glucocorticoid resistance in thymocytes from mice expressing a T cell receptor transgene.

A majority of thymocytes undergo apoptosis during differentiation due to lack of survival signals provided by T cell receptor (TCR) activation. As glucocorticoids (GC) have been suggested to be involved in this process, we have investigated the GC sensitivity in thymocytes from mice expressing a transgenic selecting TCR. We now report that immature CD4(+)CD8(+) double-positive thymocytes from these mice are comparatively more resistant to corticosterone-induced apoptosis. This is associated with reduced glucocorticoid receptor (GR) expression, increased levels of membrane CD28, increased NF-kappaB DNA binding activity, and increased binding to the CD28 response element in the interleukin-2 gene promoter. Analysis of NF-kappaB/Rel proteins from nuclear extracts demonstrated altered levels of some of these proteins. Our results suggest that TCR recognition of self major histocompatibility antigens generates intracellular signals which alter the thymocyte GC sensitivity and thereby protect them against apoptosis induced by endogenous GC.

Animals↗

PubMed2000

BRCA1 is associated with a human SWI/SNF-related complex: linking chromatin remodeling to breast cancer.

Germline mutations in the tumor suppressor gene, BRCA1, predispose individuals to breast and ovarian cancers. Using a combination of affinity- and conventional chromatographic techniques, we have isolated a predominant form of a multiprotein BRCA1-containing complex from human cells displaying chromatin-remodeling activity. Mass spectrometric sequencing of components of this complex indicated that BRCA1 is associated with a SWI/SNF-related complex. We show that BRCA1 can directly interact with the BRG1 subunit of the SWI/SNF complex. Moreover, p53-mediated stimulation of transcription by BRCA1 was completely abrogated by either a dominant-negative mutant of BRG1 or the cancer-causing deletion in exon 11 of BRCA1. These findings reveal a direct function for BRCA1 in transcriptional control through modulation of chromatin structure.

BRCA1 Protein↗

PubMed2000

Cell kinetics and differentiation after hormonal-induced prostatic hyperplasia in the dog.

BACKGROUND: Our aim was to characterize the immunophenotypical changes in canine prostate epithelium after hormonal-induced benign prostatic hyperplasia (BPH). METHODS: Castrated dogs (aged 1-2 and 9-12 years) were treated with vehicle (group C), androstanediol (group A), or androstanediol plus estradiol (group AE). Surgical prostate biopsies were obtained before and after castration and after hormonal treatment. Tissue sections were stained using antibodies specific for basal cells (34betaE12), transiently proliferating (TP)/amplifying cells (RCK103), and luminal exocrine cells (RGE53). RESULTS: Castration resulted in a marked reduction in specific immunoreactivity associated with luminal secretory cells and basal cells in young dogs. In older dogs the number of basal cells remained constant. Hormonal treatment (AE) resulted in an increased number of cells with an immunophenotype that was associated with the TP/amplifying cell compartment and hyperplastic luminal epithelium. CONCLUSIONS: The relative increase in TP/amplifying cells in hormonally induced BPH in the dog is in line with a stem-cell-derived proliferation. Moreover, the finding of androgen-independent basal cells in the prostate of older dogs may contribute to the enhanced risk of development of BPH with increasing age.

Age Factors↗

PubMed2000

Notch signaling is essential for vascular morphogenesis in mice.

The Notch gene family encodes large transmembrane receptors that are components of an evolutionarily conserved intercellular signaling mechanism. To assess the role of the Notch4 gene, we generated Notch4-deficient mice by gene targeting. Embryos homozygous for this mutation developed normally, and homozygous mutant adults were viable and fertile. However, the Notch4 mutation displayed genetic interactions with a targeted mutation of the related Notch1 gene. Embryos homozygous for mutations of both the Notch4 and Notch1 genes often displayed a more severe phenotype than Notch1 homozygous mutant embryos. Both Notch1 mutant and Notch1/Notch4 double mutant embryos displayed severe defects in angiogenic vascular remodeling. Analysis of the expression patterns of genes encoding ligands for Notch family receptors indicated that only the Dll4 gene is expressed in a pattern consistent with that expected for a gene encoding a ligand for the Notch1 and Notch4 receptors in the early embryonic vasculature. These results reveal an essential role for the Notch signaling pathway in regulating embryonic vascular morphogenesis and remodeling, and indicate that whereas the Notch4 gene is not essential during embryonic development, the Notch4 and Notch1 genes have partially overlapping roles during embryogenesis in mice.

Age Factors↗

PubMed2000

[Effect of calcitonin on the proliferation and membrane fluidity of human osteoblast-like cells].

The effects of calcitonin (CT) on the proliferation and membrane fluidity of human osteoblast-like cells line OS-732 were compared with the effect of 1,25(OH)2D3. The rate of DNA synthesis, cell proliferation, cell cycle and membrane fluidity of OS-732 cells were observed. Results showed that 1IU/ml CT stimulated cell proliferation and DNA synthesis. The percentage of cell in the G2 + M phase was increased and the percentage of cell in the synthetic phase was decreased. The membrane fluidity of OS-732 cells was increased after 0.1 mumol/L and 0.01 mumol/L 1,25(OH)2D3 treatment and was decreased after 1IU/ml CT treatment. It is concluded that calcitonin might increase the cell proliferation and decrease the membrane fluidity.

Bone Neoplasms↗

PubMed2000

Two cases of AML (M2) with a t(8;19)(q22;q13): a new cytogenetic variant.

"Simple" variants of the t(8;21) translocation involving chromosome 8 and a chromosome other than number 21 are rare. To our knowledge, only t(3;8)(q29;q22), t(8;11)(q22;q13), t(8;16)(q22;q24), t(8;20)(q22;p13), and t(8;22) have been reported in the literature. This paper describes for the first time two patients with acute myelogenous leukemia with a consistent t(8;19)(q22;q13) translocation. Their myelograms were compatible with the FAB-M2 subtype. The blasts from case 2 expressed CD34, CD33, CD13, and CD19. Karyotype analyses were performed on bone marrow cells using R- and G-banding at presentation. A t(8;19)(q22;q13) translocation was found in 28/30 metaphases for case 1 and in 23/25 metaphases for case 2. The latter case also had a deletion of chromosome 9, del(9)(q12q22) as an additional abnormality. Reverse transcriptase-polymerase chain reaction study revealed no AML1/ETO fusion transcript in case 2. Dual-color fluorescence in situ hybridization (FISH) assay using two probes (BAC92 and YAC412A4) convincingly demonstrated that the chromosomal material from 8q was translocated onto 19q rather than 19p in case 2. Thus, we consider t(8;19)(q22;q13) a true "simple" variant of t(8;21), and assume that a fusion gene resulting from the t(8;19) may contain the ETO gene located at 8q22 and an unknown partner gene from 19q13, which probably is a new transcription factor, whose molecular entity warrants further study.

Adolescent↗

PubMed2000

Genetic architecture of the polyketide synthases for methymycin and pikromycin series macrolides.

The methymycin and pikromycin series of antibiotics are structurally related macrolides produced by several Streptomyces species, including Streptomyces venezuelae ATCC 15439, which produces both 12-membered ring macrolides methymycin, neomethymycin, and 14-membered ring macrolides pikromycin and narbomycin. Cloning and sequencing of the biosynthetic gene clusters for these macrolides from three selected Streptomyces strains revealed a common genetic architecture of their polyketide synthases (PKSs). Unlike PKS clusters of other 14-membered ring macrolides such as erythromycin and oleandomycin, each of the pikromycin series producers harbors a six module PKS cluster, in which modules 5 and 6 are encoded on two separate proteins instead of one bimodular protein, as well as a thioesterase II gene immediately downstream of the main PKS gene. The results shed new light on the evolution of modular PKSs and provide further evidence on the regulation of methymycin and pikromycin production in S. venezuelae ATCC 15439.

Amino Acid Sequence↗

PubMed2000

Density functional theory study on fundamental vibrational spectra of disilyl iodide and its isotopomer.

Density function theory calculations using B3' exchange functional and LYP' correlation functionals (B3LYP) with the 3-21G** basis set were carried out to study the molecular structure and fundamental vibrational frequencies of Si2H5Br, Si2H5I and their isotopomers. One scale factor used to scale Si-H(D) force constants for Si2H5Br is transferred from Si2H5Br to Si2H5I. Other scale factors of force constants for Si2H5I and Si2D5I are determined by a least-squares fitting to experimental vibrational frequencies. The predictions of fundamental vibrational frequencies for Si2H5I and its isotopomer are given. The scaled DFT force fields using B3LYP/3-21G** reproduced the observed fundamental vibrational frequencies of Si2H5I and its deuterium isotopomer with a mean absolute deviation of 6 cm(-1). Reassignment of some fundamental vibrational modes of Si2H5I and its isotopomer is discussed.

Silanes↗

PubMed2000

Alternative modular polyketide synthase expression controls macrolactone structure.

Modular polyketide synthases are giant multifunctional enzymes that catalyse the condensation of small carboxylic acids such as acetate and propionate into structurally diverse polyketides that possess a spectrum of biological activities. In a modular polyketide synthase, an enzymatic domain catalyses a specific reaction, and three to six enzymatic domains involved in a condensation-processing cycle are organized into a module. A fundamental aspect of a modular polyketide synthase is that its module arrangement linearly specifies the structure of its polyketide product. Here we report a natural example in which alternative expression of the pikromycin polyketide synthase results in the generation of two macrolactone structures. Expression of the full-length modular polyketide synthase PikAIV in Streptomyces venezuelae generates the 14-membered ring macrolactone narbonolide, whereas expression of the amino-terminal truncated form of PikAIV leads to 'skipping' of the final condensation cycle in polyketide biosynthesis to generate the 12-membered ring macrolactone 10-deoxymethynolide. Our findings provide insight into the structure and function of modular polyketide synthases, as well as a new set of tools to generate structural diversity in polyketide natural products.

Anti-Bacterial Agents↗

PubMed2000

Neuroendocrine cells during human prostate development: does neuroendocrine cell density remain constant during fetal as well as postnatal life?

BACKGROUND: Knowledge concerning differentiation of neuroendocrine (NE) cells during development of the human prostate is rather fragmentary. Using immunohistochemistry combined with a morphometric method, we investigated the distribution and density of NE cells in the developing human prostate, with special emphasis on the topographical relationship of NE cells with the developing gland. METHODS: Consecutive sections from a total of 42 human prostates taken during autopsy of fetuses (12-38 weeks of gestation), prepubertal males, and young adults were immunostained for chromogranin A and serotonin. Computer-assisted image analysis was used to assess the total number of cells in the different parts of the branching glandular anlage, i.e., budding tips and acini/ducts. Next, the number of NE cells was counted manually. The NE cell density (NE cell index) was then determined. RESULTS: NE cells could first be detected in the prostate from 13 weeks of gestation. By 21 weeks of gestation, all prostates contained NE cells. NE cells were mainly confined to the acinous/ductal regions, while most of the budding tips lacked NE staining. NE cell indexes of individuals were highly variable, mostly in the youngest age group. CONCLUSIONS: In the normal prostate, NE cell density probably remains constant in acini/ducts from fetuses to young adulthood. The presence of neuroendocrine cells in well-developed glandular structures at such an early fetal age and their absence in the less differentiated budding tips possibly indicates that differentiation of NE cells is associated with glandular maturation. NE cells occur preferentially in the acinous/ductal region, implying a paracrine function during secretory differentiation of exocrine epithelial cells.

Adolescent↗

PubMed2000

Mechanisms of molecular recognition in the pikromycin polyketide synthase.

BACKGROUND: Modular polyketide synthases (PKSs) produce a wide range of medically significant compounds. In the case of the pikromycin PKS of Streptomyces venezuelae, four separate polypeptides (PikAI-PikAIV), comprising a total of one loading domain and six extension modules, generate the 14-membered ring macrolactone narbonolide. The polypeptide PikAIV contains a thioesterase (TE) domain and is responsible for catalyzing both the last elongation step with methylmalonyl CoA, and subsequent release of the final polyketide chain elongation intermediate from the PKS. Under certain growth conditions this polypeptide is synthesized from an alternative translational start site, giving rise to an N-terminal truncated form of PikAIV, containing only half of the ketosynthase (KS(6)) domain. The truncated form of PikAIV is unable to catalyze the final elongation step, but is able to cleave a polyketide chain from the preceding module on PikAIII (ACP(5)), giving rise to the 12-membered ring product 10-deoxymethynolide. RESULTS: S. venezuelae mutants expressing hybrid PikAIV polypeptides containing acyl carrier protein (ACP) and malonyl CoA specific acyltransferase (AT) domains from the rapamycin PKS were unable to catalyze production of 12- or 14-membered ring macrolactone products. Plasmid-based expression of a hybrid PikAIV containing the native KS(6) and TE domains, however, restored production of both narbonolide and 10-deoxymethynolide in the S. venezuelae AX912 mutant that generates a TE-deleted form of PikAIV. Use of alternative KS domains or deletion of the KS(6) domain within the hybrid PikAIV resulted in loss of both products. Plasmid-based expression of a TE domain of PikAIV as a separate polypeptide in the AX912 mutant resulted in greater than 50% restoration of 10-deoxymethynolide, but not in mutants expressing a hybrid PikAIV bearing an unnatural AT domain. Mutants expressing hybrid PikAIV polypeptides containing the natural AT(6) domains and different ACP domains efficiently produced polyketide products, but with a significantly higher 10-deoxymethynolide/narbonolide ratio than observed with native PikAIV. CONCLUSIONS: Dimerization of KS(6) modules allows in vivo formation of a PKS heterodimer using PikAIV polypeptides containing different AT and ACP domains. In such heterodimers, the TE domain and the AT(6) domain responsible for formation of the narbonolide product are located on different polypeptide chains. The AT(6) domain of PikAIV plays an important role in facilitating TE-catalyzed chain termination (10-deoxymethynolide formation) at the proceeding module in PikAIII. The pikromycin PKS can tolerate the presence of multiple forms (active and inactive) of PikAIV, and decreased efficiency of elongation by PikAIV can result in increased levels of 10-deoxymethynolide. These results provide new insight into functional molecular interactions and interdomain recognition in modular PKSs.

Anti-Bacterial Agents↗

PubMed2000

Density functional theory studies on tautomeric stability and infrared spectra of 2-chloroadenine.

The tautomeric stability and vibrational IR spectrum of 2-chloroadenine were studied using density functional theory (DFT) at B3LYP/6-31G* level. The amino N(9)H tautomer of 2-chloroadenine was predicted to be most stable. A scaled quantum mechanical (SQM) force field approach was used to calculate the vibrational frequencies of amino N(9)H form of 2-chloroadenine. The force constant scale factors were transferred from those of purine. The mean deviation between the predicted vibrational frequencies and the observed ones is 8.0 cm(-1). The results made it possible to give complete assignments of the IR spectrum of this molecule.

Adenine↗

PubMed2000

Anti-CD3 induced thymocyte apoptosis in vivo require the antibody Fc domain.

We have earlier found that explanted thymic epithelial cells (TEC) can produce glucocorticoid (GC) activity in vitro and that the GC receptor (GR) antagonist RU486 partially inhibit thymic apoptosis induced by the anti-CD3 monoclonal antibodies (MoAb) 2C11, both in vivo and in new-born thymic organ cultures. To explain the inhibitory effect of RU486 in this system we have now investigated the importance of the 2C11 Fc as this MoAb bind with high affinity to cellular FcR. We have found both that whole 2C11 MoAb can bind to explanted TEC in vitro and that F(ab)'2 fragments from this MoAb loose this ability, in addition with the capacity to induce thymic apoptosis in vivo. We interpret our results to indicate that the injected 2C11 MoAb may establish a close contact between GC producing, FcR positive TEC cells and CD3 positive thymocytes and thereby subject the later to high paracrine GC concentrations and subsequent induction of apoptosis.

Animals↗

PubMed2000

The G protein-coupled receptor gpr1 is a nutrient sensor that regulates pseudohyphal differentiation in Saccharomyces cerevisiae.

Pseudohyphal differentiation in the budding yeast Saccharomyces cerevisiae is induced in diploid cells in response to nitrogen starvation and abundant fermentable carbon source. Filamentous growth requires at least two signaling pathways: the pheromone responsive MAP kinase cascade and the Gpa2p-cAMP-PKA signaling pathway. Recent studies have established a physical and functional link between the Galpha protein Gpa2 and the G protein-coupled receptor homolog Gpr1. We report here that the Gpr1 receptor is required for filamentous and haploid invasive growth and regulates expression of the cell surface flocculin Flo11. Epistasis analysis supports a model in which the Gpr1 receptor regulates pseudohyphal growth via the Gpa2p-cAMP-PKA pathway and independently of both the MAP kinase cascade and the PKA related kinase Sch9. Genetic and physiological studies indicate that the Gpr1 receptor is activated by glucose and other structurally related sugars. Because expression of the GPR1 gene is known to be induced by nitrogen starvation, the Gpr1 receptor may serve as a dual sensor of abundant carbon source (sugar ligand) and nitrogen starvation. In summary, our studies reveal a novel G protein-coupled receptor senses nutrients and regulates the dimorphic transition to filamentous growth via a Galpha protein-cAMP-PKA signal transduction cascade.

Carbohydrate Metabolism↗

PubMed2000

Saccharomyces cerevisiae RAI1 (YGL246c) is homologous to human DOM3Z and encodes a protein that binds the nuclear exoribonuclease Rat1p.

The RAT1 gene of Saccharomyces cerevisiae encodes a 5'-->3' exoribonuclease which plays an essential role in yeast RNA degradation and/or processing in the nucleus. We have cloned a previously uncharacterized gene (YGL246c) that we refer to as RAI1 (Rat1p interacting protein 1). RAI1 is homologous to Caenorhabditis elegans DOM-3 and human DOM3Z. Deletion of RAI1 confers a growth defect which can be complemented by an additional copy of RAT1 on a centromeric vector or by directing Xrn1p, the cytoplasmic homolog of Rat1p, to the nucleus through the addition of a nuclear targeting sequence. Deletion of RAI1 is synthetically lethal with the rat1-1(ts) mutation and shows genetic interaction with a deletion of SKI2 but not XRN1. Polysome analysis of an rai1 deletion mutant indicated a defect in 60S biogenesis which was nearly fully reversed by high-copy RAT1. Northern blot analysis of rRNAs revealed that rai1 is required for normal 5.8S processing. In the absence of RAI1, 5.8S(L) was the predominant form of 5.8S and there was an accumulation of 3'-extended forms but not 5'-extended species of 5. 8S. In addition, a 27S pre-rRNA species accumulated in the rai1 mutant. Thus, deletion of RAI1 affects both 5' and 3' processing reactions of 5.8S rRNA. Consistent with the in vivo data suggesting that RAI1 enhances RAT1 function, purified Rai1p stabilized the in vitro exoribonuclease activity of Rat1p.

Amino Acid Sequence↗