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Biomedical subjects

K Ye

Publications and source records attributed to K Ye.

At least 19 recordsLinked to original sources
PubMed2006

Src-family tyrosine kinase fyn phosphorylates phosphatidylinositol 3-kinase enhancer-activating Akt, preventing its apoptotic cleavage and promoting cell survival.

Phosphatidylinositol 3-kinase enhancer-activating Akt (PIKE-A) binds Akt and upregulates its kinase activity, preventing apoptosis. PIKE-A can be potently phosphorylated on tyrosine residues 682 and 774, leading to its resistance to caspase cleavage. However, the upstream tyrosine kinases responsible for PIKE-A phosphorylation and subsequent physiological significance remain unknown. Here, we show that PIKE-A can be cleaved by the active apoptosome at both D474 and D592 residues. Employing fyn-deficient mouse embryonic fibroblast cells and tissues, we demonstrate that fyn is essential for phosphorylating PIKE-A and protects it from apoptotic cleavage. Active but not kinase-dead fyn interacts with PIKE-A and phosphorylates it on both Y682 and Y774 residues. Tyrosine phosphorylation in PIKE-A is required for its association with active fyn but not for Akt. Mutation of D into A in PIKE-A protects it from caspase cleavage and promotes cell survival. Thus, this finding provides a molecular mechanism accounting for the antiapoptotic action of src-family tyrosine kinase.

Animals↗
PubMed2003

A model to estimate the optimal sample size for microbiological surveys.

Estimating optimal sample size for microbiological surveys is a challenge for laboratory managers. When insufficient sampling is conducted, biased inferences are likely; however, when excessive sampling is conducted valuable laboratory resources are wasted. This report presents a statistical model for the estimation of the sample size appropriate for the accurate identification of the bacterial subtypes of interest in a specimen. This applied model for microbiology laboratory use is based on a Bayesian mode of inference, which combines two inputs: (ii) a prespecified estimate, or prior distribution statement, based on available scientific knowledge and (ii) observed data. The specific inputs for the model are a prior distribution statement of the number of strains per specimen provided by an informed microbiologist and data from a microbiological survey indicating the number of strains per specimen. The model output is an updated probability distribution of strains per specimen, which can be used to estimate the probability of observing all strains present according to the number of colonies that are sampled. In this report two scenarios that illustrate the use of the model to estimate bacterial colony sample size requirements are presented. In the first scenario, bacterial colony sample size is estimated to correctly identify Campylobacter amplified restriction fragment length polymorphism types on broiler carcasses. The second scenario estimates bacterial colony sample size to correctly identify Salmonella enterica serotype Enteritidis phage types in fecal drag swabs from egg-laying poultry flocks. An advantage of the model is that as updated inputs from ongoing surveys are incorporated into the model, increasingly precise sample size estimates are likely to be made.

Animals↗
PubMed2001

Sustained activation of p34(cdc2) is required for noscapine-induced apoptosis.

Mitotic arrest and subsequent apoptosis has been observed in many types of cells treated with anti-microtubule agents. However, the molecular mechanisms underlying the two events as well as their relationship are not well understood; on the contrary, there has been increasing evidence indicating that anti-microtubule agents might induce apoptosis via signaling pathways independent of mitosis. In this study, we found that apoptosis induced by noscapine, an anti-microtubule drug previously shown to cause both mitotic arrest and apoptotic cell death, was blocked by inhibiting p34(cdc2) activity with olomoucine in FM3A murine mammary carcinoma cells or by reducing the level and activity of p34(cdc2) in a mutant cell line FT210 derived from FM3A. Furthermore, transfection of the mutant FT210 cells with wild-type p34(cdc2) restored their ability to undergo mitotic arrest and then apoptosis in response to noscapine. Thus, we conclude that sustained activation of the p34(cdc2) kinase during mitotic arrest is required for subsequent apoptosis induced by noscapine, establishing a link between the two events.

Animals↗
PubMed2001

GRAB: a physiologic guanine nucleotide exchange factor for Rab3A, which interacts with inositol hexakisphosphate kinase.

Diphosphoinositol-pentakisphosphate (InsP7) and bis-diphosphoinositol tetrakisphosphate (InsP8) possess pyrophosphate bonds. InsP7 is formed from inositol hexakisphosphate (InsP6) by recently identified InsP6 kinases designated InsP6K1 and InsP6K2. We now report the identification, cloning, and characterization of a novel protein, GRAB (guanine nucleotide exchange factor for Rab3A), which interacts with both InsP6K1 and Rab3A, a Ras-like GTPase that regulates synaptic vesicle exocytosis. GRAB is a physiologic GEF (guanine nucleotide exchange factor) for Rab3A. Consistent with a role of Rab3A in synaptic vesicle exocytosis, GRAB regulates depolarization-induced release of dopamine from PC12 cells and nicotinic agonist-induced hGH release from bovine adrenal chromaffin cells. The association of InsP6K1 with GRAB fits with a role for InsP7 in vesicle exocytosis.

Adrenal Medulla↗
PubMed2001

Self-association reaction of denatured staphylococcal nuclease fragments characterized by heteronuclear NMR.

The self-association reaction of denatured staphylococcal nuclease fragments, urea-denatured G88W110, containing residues 1-110 and mutation G88W, and physiologically denatured 131-residue Delta 131 Delta, have been characterized by NMR at close to neutral pH. The two fragments differ in the extent and degree of association due to the different sequence and experimental conditions. Residues 13-39, which show significant exchange line broadening, constitute the main association interface in both fragments. A second weak association region was identified involving residues 79-105 only in the case of urea-denatured G88W110. For residues involved in the association reaction, significant suppression of the line broadening and small but systematic chemical shift variation of the amide protons were observed as the protein concentration decreased. The direction of chemical shift change suggests that the associated state adopts mainly beta-sheet-like conformation, and the beta-hairpin formed by strands beta 2 and beta 3 is native-like. The apparent molecular size obtained by diffusion coefficient measurements shows a weak degree of association for Delta 131 Delta below 0.4 mM protein concentration and for G88W110 in 4 M urea. In both cases the fragments are predominantly in the monomeric state. However, the weak association reaction can significantly influence the transverse relaxation of residues involved in the association reaction. The degree of association abruptly increases for Delta 131 Delta above 0.4 mM concentration, and it is estimated to form a 4 to 8 mer at 2 mM. It is proposed that the main region involved in association forms the core structure, with the remainder of residues largely disordered in the associated state. Despite the obvious influence of the association reaction on the slow motion of the backbone, the restricted mobility on the nanosecond timescale around the region of strand beta 5 is essentially unaffected by the association reaction and degree of denaturation.

Magnetic Resonance Spectroscopy↗
PubMed2001

FKBP12, the 12-kDa FK506-binding protein, is a physiologic regulator of the cell cycle.

FKBP12, the 12-kDa FK506-binding protein, is a ubiquitous abundant protein that acts as a receptor for the immunosuppressant drug FK506, binds tightly to intracellular calcium release channels and to the transforming growth factor beta (TGF-beta) type I receptor. We now demonstrate that cells from FKBP12-deficient (FKBP12(-/-)) mice manifest cell cycle arrest in G(1) phase and that these cells can be rescued by FKBP12 transfection. This arrest is mediated by marked augmentation of p21(WAF1/CIP1) levels, which cannot be further augmented by TGF-beta1. The p21 up-regulation and cell cycle arrest derive from the overactivity of TGF-beta receptor signaling, which is normally inhibited by FKBP12. Cell cycle arrest is prevented by transfection with a dominant-negative TGF-beta receptor construct. TGF-beta receptor signaling to gene expression can be mediated by SMAD, p38, and ERK/MAP kinase (extracellular signal-regulated kinase/mitogen-activated protein kinase) pathways. SMAD signaling is down-regulated in FKBP12(-/-) cells. Inhibition of ERK/MAP kinase fails to affect p21 up-regulation. By contrast, activated phosphorylated p38 is markedly augmented in FKBP12(-/-) cells and the p21 up-regulation is prevented by an inhibitor of p38. Thus, FKBP12 is a physiologic regulator of cell cycle acting by normally down-regulating TGF-beta receptor signaling.

Animals↗
PubMed2001

Statistical assessment of violations of water quality standards under Section 303(d) of the clean water act.

Section 303(d) of the Clean Water Act requires states to assess the condition of their waters and to implement plans to improve the quality of waters identified as impaired. U.S. Environmental Protection Agency guidelines require a stream segment to be listed as impaired when greater than 10% of the measurements of water quality conditions exceed numeric criteria. This can be termed a "raw score" assessment approach. Water quality measurements are samples taken from a population of water quality conditions. Concentrations of pollutants vary naturally, measurement errors may be made, and occasional violations of a standard may be tolerable. Therefore, it is reasonable to view the assessment process as a statistical decision problem. Assessment of water quality conditions must be cognizant of the possibility of type I (a false declaration of standards violation) and type II (a false declaration of no violation) errors. The raw score approach is shown to have a high type I error rate. Alternatives to the raw score approach are the Binomial test and the Bayesian Binomial approach. These methods use the same information to make decisions but allow for control of the error rates. The two statistical methods differ based on consideration of prior information about violation. Falsely concluding that a water segment is impaired results in unnecessary planning and pollution control implementation costs. On the other hand, falsely concluding that a segment is not impaired may pose a risk to human health or to the services of the aquatic environment. An approach that recognizes type I and type II error in the water quality assessment process is suggested.

Bayes Theorem↗
PubMed2001

Creation of cell surface-engineered yeast that display different fluorescent proteins in response to the glucose concentration.

We have successfully created a novel yeast strain able to monitor changes in environmental conditions by displaying either green fluorescent protein (GFP) from Aequorea victoria or blue fluorescent protein (BFP), a variant of GFP, on its cell surface as a visible reporter. For the display of these fluorescent proteins on the cell surface of Saccharomyces cerevisiase, our cell-surface-engineering system was utilized. The GAPDH promoter, which is active in the presence of glucose, and the UPR-ICL promoter from Candida tropicalis, which starts to function in the presence of a reduced level of glucose, were employed simultaneously to express the GFP-encoding gene and the BFP-encoding gene, respectively. This cell-surface-engineered yeast strain emitted green fluorescence from the cell surface when sufficient glucose was present in the medium, and blue fluorescence from the same cell surface when the glucose in the medium was consumed. The fluorescent proteins displayed on the cell surface using the different promoters enabled us to monitor the concentrations of intra- and/or extracellular glucose that regulated activation or inactivation of the promoters. This novel yeast strain could facilitate the computerized control of various bioprocesses measuring emitted fluorescence.

Genetic Engineering↗
PubMed2001

Effects of charged amino-acid mutation on the solution structure of cytochrome b(5) and binding between cytochrome b(5) and cytochrome c.

The solution structure of oxidized bovine microsomal cytochrome b(5) mutant (E48, E56/A, D60/A) has been determined through 1524 meaningful nuclear Overhauser effect constraints together with 190 pseudocontact shift constraints. The final family of 35 conformers has rmsd values with respect to the mean structure of 0.045+/-0.009 nm and 0.088+/-0.011 nm for backbone and heavy atoms, respectively. A characteristic of this mutant is that of having no significant changes in the whole folding and secondary structure compared with the X-ray and solution structures of wild-type cytochrome b(5). The binding of different surface mutants of cytochrome b(5) with cytochrome c shows that electrostatic interactions play an important role in maintaining the stability and specificity of the protein complex formed. The differences in association constants demonstrate the electrostatic contributions of cytochrome b(5) surface negatively charged residues, which were suggested to be involved in complex formation in the Northrup and Salemme models, have cumulative effect on the stability of cyt c-cyt b(5) complex, and the contribution of Glu48 is a little higher than that of Glu44. Moreover, our result suggests that the docking geometry proposed by Northrup, which is involved in the participation of Glu48, Glu56, Asp60, and heme propionate of cytochrome b(5), do occur in the association between cytochrome b(5) and cytochrome c.

Amino Acid Sequence↗
PubMed2000

Pike. A nuclear gtpase that enhances PI3kinase activity and is regulated by protein 4.1N.

While cytoplasmic PI3Kinase (PI3K) is well characterized, regulation of nuclear PI3K has been obscure. A novel protein, PIKE (PI3Kinase Enhancer), interacts with nuclear PI3K to stimulate its lipid kinase activity. PIKE encodes a 753 amino acid nuclear GTPase. Dominant-negative PIKE prevents the NGF enhancement of PI3K and upregulation of cyclin D1. NGF treatment also leads to PIKE interactions with 4.1N, which has translocated to the nucleus, fitting with the initial identification of PIKE based on its binding 4.1N in a yeast two-hybrid screen. Overexpression of 4.1N abolishes PIKE effects on PI3K. Activation of nuclear PI3K by PIKE is inhibited by the NGF-stimulated 4.1N translocation to the nucleus. Thus, PIKE physiologically modulates the activation by NGF of nuclear PI3K.

Adenosine Triphosphate↗
PubMed2000

Improvement of vitamin B(12) fermentation by reducing the inhibitory metabolites by cell recycle system and a mixed culture.

The major problem in vitamin B(12) production using Propionibacterium is the growth inhibition of the cell due to the accumulation of inhibitory metabolites such as propionic acid and acetic acid. In the present paper, we considered several approaches of controlling the propionic acid concentration at low level. Namely: (1) the periodic cultivation of Propionibacterium where dissolved oxygen (DO) concentration was alternatively changed between 0 and 1ppm; (2) cell recycle system using hollow fiber module; and (3) mixed culture using Propionibacterium and Ralstonia eutropha where the latter microorganism assimilates the propionic acid produced by the former. It was found that the productivity of vitamin B(12) was the highest for the cell recycle system, while if the performance was evaluated based on the amount of vitamin B(12) produced per medium used, the mixed culture system gave the far highest value.

Journal Article↗
PubMed2000

The possible subduction of continental material to depths greater than 200 km.

Determining the depth to which continental lithosphere can be subducted into the mantle at convergent plate boundaries is of importance for understanding the long-term growth of supercontinents as well as the dynamic processes that shape such margins. Recent discoveries of coesite and diamond in regional ultrahigh-pressure (UHP) metamorphic rocks has demonstrated that continental material can be subducted to depths of at least 120 km (ref. 1), and subduction to depths of 150-300 km has been inferred from garnet peridotites in orogenic UHP belts based on several indirect observations. But continental subduction to such depths is difficult to trace directly in natural UHP metamorphic crustal rocks by conventional mineralogical and petrological methods because of extensive late-stage recrystallization and the lack of a suitable pressure indicator. It has been predicted from experimental work, however, that solid-state dissolution of pyroxene should occur in garnet at depths greater than 150 km (refs 6-8). Here we report the observation of high concentrations of clinopyroxene, rutile and apatite exsolutions in garnet within eclogites from Yangkou in the Sulu UHP metamorphic belt, China. We interpret these data as resulting from the high-pressure formation of pyroxene solid solutions in subducted continental material. Appropriate conditions for the Na2O concentrations and octahedral silicon observed in these samples are met at depths greater than 200 km.

Journal Article↗
PubMed2000

Interactions between subdomains in the partially folded state of staphylococcal nuclease.

Staphylococcal nuclease can be roughly divided into a beta-subdomain in N-terminal and an alpha-subdomain in C-terminal. They fold sequentially under certain conditions, causing a partially folded intermediate state in which the native-like beta-barrel persists while alpha-helix regions largely disorder. To investigate the possible long-range interactions between the two subdomains in the intermediate, N-terminal fragments have been used as intermediate analogues, with polypeptide ending at positions 102, 110, 121 and 135 and with a tryptophan substitution at position 66 or 88 to facilitate the observation of the beta-barrel. Segment-resolved interactions between beta-barrel and residues 103-135 were identified by comparing their spectroscopic properties of fluorescence, circular dichroism and NMR and by their stability. Except for unstable V66W102, the guanidine and thermal denaturation of fragments are cooperative and well approximated by the two-state transition. Minimal stable structure units of both tryptophan-containing fragments comprise residues 1-110. With the main interaction in segment 103-135, residues 103-110 contribute approximate 2 kcal/mol to the stability. Elongation of C-terminal from 110 residue neither increases the stability nor alters the structure core of the G88W fragments. However, residues 111-121 influence the tertiary structure of the V66W fragments suggesting its minor interactions with beta-barrel.

Circular Dichroism↗
PubMed2000

Construction of an engineered yeast with glucose-inducible emission of green fluorescence from the cell surface.

An engineered yeast with emission of fluorescence from the cell surface was constructed. Cell surface engineering was applied to display a visible reporter molecule, green fluorescent protein (GFP). A glucose-inducible promoter GAPDH as a model promoter was selected to control the expression of the reporter gene in response to environmental changes. The GFP gene was fused with the gene encoding the C-terminal half of alpha-agglutinin of Saccharomyces cerevisiae having a glycosylphosphatidylinositol anchor attachment signal sequence. A secretion signal sequence of the fungal glucoamylase precursor protein was connected to the N-terminal of GFP. This designed gene was integrated into the TRP1 locus of the chromosome of S. cerevisiae with homologous recombination. Fluorescence microscopy demonstrated that the transformant cells emitted green fluorescence derived from functionally expressed GFP involved in the fusion molecule. The surface display of GFP was further verified by immunofluorescence labeling with a polyclonal antibody (raised in rabbits) against GFP as the first antibody and Rhodamine Red-X-conjugated goat anti-rabbit IgG as the second antibody which cannot penetrate into the cell membrane. The display of GFP on the cell surface was confirmed using a confocal laser scanning microscope and by measuring fluorescence in each cell fraction obtained after the subcellular fractionation. As GFP was proved to be displayed as an active form on the cell surface, selection of promoters will endow yeast cells with abilities to respond to changes in environmental conditions, including nutrient concentrations in the media, through the emission of fluorescence.

Base Sequence↗
PubMed2000

Noscapine inhibits tumor growth with little toxicity to normal tissues or inhibition of immune responses.

Noscapine, a phthalideisoquinoline alkaloid derived from opium, has been used as an oral anti-tussive agent and has shown very few toxic effects in animals or humans. Recently, we reported that noscapine binds stoichiometrically to tubulin and promotes microtubule polymerization. Noscapine causes growth arrest of tumor cells in mitosis and induces apoptosis of tumor cells in vitro. Previous experiments also showed that noscapine has potent antitumor activity in mice when administered parenterally or by gastric lavage. Here, we report that the anti-mitotic effect was specific to noscapine since closely related compounds did not inhibit the growth of a lymphoma cell line. In addition, noscapine was shown to be effective in reducing the growth of the lymphoma and increasing the survival of tumor-bearing mice when administered in the drinking water. It is noteworthy that, noscapine showed little or no toxicity to kidney, liver, heart, bone marrow, spleen or small intestine at tumor-suppressive doses. Furthermore, oral noscapine did not inhibit primary immune responses, which are critically dependent upon proliferation of lymphoid cells. Thus, our results indicate that noscapine has the potential to be an effective chemotherapeutic agent for the treatment of human cancer.

Alkaloids↗
PubMed2000

Dexras1: a G protein specifically coupled to neuronal nitric oxide synthase via CAPON.

Because nitric oxide (NO) is a highly reactive signaling molecule, chemical inactivation by reaction with oxygen, superoxide, and glutathione competes with specific interactions with target proteins. NO signaling may be enhanced by adaptor proteins that couple neuronal NO synthase (nNOS) to specific target proteins. Here we identify a selective interaction of the nNOS adaptor protein CAPON with Dexras1, a brain-enriched member of the Ras family of small monomeric G proteins. We find that Dexras1 is activated by NO donors as well as by NMDA receptor-stimulated NO synthesis in cortical neurons. The importance of Dexras1 as a physiologic target of nNOS is established by the selective decrease of Dexras1 activation, but not H-Ras or four other Ras family members, in the brains of mice harboring a targeted genomic deletion of nNOS (nNOS-/-). We also find that nNOS, CAPON, and Dexras1 form a ternary complex that enhances the ability of nNOS to activate Dexras1. These findings identify Dexras1 as a novel physiologic NO effector and suggest that anchoring of nNOS to specific targets is a mechanism by which NO signaling is enhanced.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Evaluation of a method for experimental induction of osteoarthritis of the hip joints in dogs.

OBJECTIVE: To evaluate a method for experimental induction of osteoarthritis in the hip joints of dogs. ANIMALS: 12 mixed-breed dogs. PROCEDURE: A unilateral triple pelvic osteotomy was performed. In 6 dogs, the iliac osteotomy was repaired with 45 degrees of internal rotation, reducing coverage of the femoral head by the acetabulum. In the other 6 dogs, the fragments were repaired in anatomic alignment. Radiography, force plate evaluations, and subjective lameness evaluations were performed before and after surgery. Dogs were euthanatized 7 months after surgery, and samples of cartilage and joint capsule were examined histologically. RESULTS: Subjective lameness scores, radiographic appearance of the hip joints, and Norberg angles were not significantly different between groups; however, force plate evaluations did reveal significant differences in vertical ground reaction forces. Femoral head coverage was significantly decreased with rotation of the acetabulum. Mild inflammatory changes were discernible in the joint capsule and articular cartilage of some dogs in both groups. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that 45 degrees internal rotation of the acetabulum does not consistently induce biologically important osteoarthritic changes in the hip joints of dogs.

Acetabulum↗