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A Yee

Publications and source records attributed to A Yee.

At least 19 recordsLinked to original sources
PubMed2005

Optimization of resolution and sensitivity of 4D NOESY using multi-dimensional decomposition.

Highly resolved multi-dimensional NOE data are essential for rapid and accurate determination of spatial protein structures such as in structural genomics projects. Four-dimensional spectra contain almost no spectral overlap inherently present in lower dimensionality spectra and are highly amenable to application of automated routines for spectral resonance location and assignment. However, a high resolution 4D data set using conventional uniform sampling usually requires unacceptably long measurement time. Recently we have reported that the use of non-uniform sampling and multi-dimensional decomposition (MDD) can remedy this problem. Here we validate accuracy and robustness of the method, and demonstrate its usefulness for fully protonated protein samples. The method was applied to 11 kDa protein PA1123 from structural genomics pipeline. A systematic evaluation of spectral reconstructions obtained using 15-100% subsets of the complete reference 4D 1H-13C-13C-1H NOESY spectrum has been performed. With the experimental time saving of up to six times, the resolution and the sensitivity per unit time is shown to be similar to that of the fully recorded spectrum. For the 30% data subset we demonstrate that the intensities in the reconstructed and reference 4D spectra correspond with a correlation coefficient of 0.997 in the full range of spectral amplitudes. Intensities of the strong, middle and weak cross-peaks correlate with coefficients 0.9997, 0.9965, and 0.83. The method does not produce false peaks. 2% of weak peaks lost in the 30% reconstruction is in line with theoretically expected noise increase for the shorter measurement time. Together with good accuracy in the relative line-widths these translate to reliable distance constrains derived from sparsely sampled, high resolution 4D NOESY data.

Fourier Analysis↗
PubMed2004

Statin use in Type 2 diabetes mellitus is associated with a delay in starting insulin.

AIMS: It has been suggested that HMG Co-A reductase inhibitors ('statins') may reduce the risk of developing Type 2 diabetes mellitus. This study was designed to evaluate whether use of statins would also delay progression to insulin therapy. METHODS: This was a retrospective cohort study using Saskatchewan Health databases to identify subjects newly started on oral antidiabetic agents from 1991 to 1996. SUBJECTS: < 30 years of age or with previous lipid-lowering drug use were excluded. Medications known to influence glycaemic control, co-morbidity, and demographic data were collected. Statin exposure was defined as at least 1 year of use. Primary outcome was starting insulin treatment. Multivariate Cox proportional hazards models were used to examine the association between statin use and starting insulin. RESULTS: The final cohort included 10,996 new users of oral antidiabetic agents, of which 484 (4.4%) used statins. Mean age was 64 years and 55% were male. Mean duration of follow-up was 5.1 years; 11.1% (n = 1221) eventually started insulin treatment. Statin users were no less likely than non-users to start insulin treatment eventually (11.6% vs. 11.1%, P = 0.74). After multivariate adjustment, however, statin use was associated with a 10-month delay before newly treated diabetic subjects needed to start insulin treatment (adjusted hazard ratio 0.74; 95% confidence interval 0.56, 0.97, P = 0.028). CONCLUSION: The use of statins is associated with a delay in starting insulin treatment in patients with Type 2 diabetes initially treated with oral antidiabetic agents. Whether this relationship exists for patients at high risk of developing diabetes should be examined in a randomized trial.

Administration, Oral↗
PubMed2001

SPINE: an integrated tracking database and data mining approach for identifying feasible targets in high-throughput structural proteomics.

High-throughput structural proteomics is expected to generate considerable amounts of data on the progress of structure determination for many proteins. For each protein this includes information about cloning, expression, purification, biophysical characterization and structure determination via NMR spectroscopy or X-ray crystallography. It will be essential to develop specifications and ontologies for standardizing this information to make it amenable to retrospective analysis. To this end we created the SPINE database and analysis system for the Northeast Structural Genomics Consortium. SPINE, which is available at bioinfo.mbb.yale.edu/nesg or nesg.org, is specifically designed to enable distributed scientific collaboration via the Internet. It was designed not just as an information repository but as an active vehicle to standardize proteomics data in a form that would enable systematic data mining. The system features an intuitive user interface for interactive retrieval and modification of expression construct data, query forms designed to track global project progress and external links to many other resources. Currently the database contains experimental data on 985 constructs, of which 740 are drawn from Methanobacterium thermoautotrophicum, 123 from Saccharomyces cerevisiae, 93 from Caenorhabditis elegans and the remainder from other organisms. We developed a comprehensive set of data mining features for each protein, including several related to experimental progress (e.g. expression level, solubility and crystallization) and 42 based on the underlying protein sequence (e.g. amino acid composition, secondary structure and occurrence of low complexity regions). We demonstrate in detail the application of a particular machine learning approach, decision trees, to the tasks of predicting a protein's solubility and propensity to crystallize based on sequence features. We are able to extract a number of key rules from our trees, in particular that soluble proteins tend to have significantly more acidic residues and fewer hydrophobic stretches than insoluble ones. One of the characteristics of proteomics data sets, currently and in the foreseeable future, is their intermediate size ( approximately 500-5000 data points). This creates a number of issues in relation to error estimation. Initially we estimate the overall error in our trees based on standard cross-validation. However, this leaves out a significant fraction of the data in model construction and does not give error estimates on individual rules. Therefore, we present alternative methods to estimate the error in particular rules.

Animals↗
PubMed2001

Diffusion and calibration properties of microdialysis sampling membranes in biological media.

The diffusion and calibration properties for three commercially available microdialysis membranes (polycarbonate-polyether (PC), polyacrylonitrile (PAN), and cuprophan (CUP)) were evaluated. The analytes studied had molecular weights between 94 (phenol) and 1355 (vitamin B12). For each analyte-membrane pair, an effective membrane diffusion coefficient was calculated. Effective membrane diffusion coefficients varied considerably between the microdialysis membranes. For Vitamin B12, CUP and PAN membranes gave relative recovery values of greater than 20% at 0.5 microl min(-1), while the PC membrane had a 1% recovery. When backpressure was applied. PC and PAN membranes exhibited more ultrafiltration than CUP membranes. Ultrafiltration did not affect analyte relative recovery through either PC or PAN membranes. Effective membrane diffusion coefficients were not significantly altered for some membrane-analyte combinations when exposed to 4% bovine serum albumin or 0.3% fibrinogen. These data suggest that reductions in relative recovery during long-term microdialysis sampling experiments may be due to other physiologically relevant proteins or to tissue reactions near the dialysis membrane.

Calibration↗
PubMed2001

Phenotypic differentiation of TGF-beta1-responsive pluripotent premesenchymal prehematopoietic progenitor (P4 stem) cells from murine bone marrow.

On the horizon of modern molecular medicine is the requisite technology to capture multipotent human stem cells that are capable of self-renewal and to direct these stem cells along defined lineages for therapeutic purposes. In this article, we describe the hematopoietic and mesenchymal differentiation potential of a unique population of transforming growth factor-beta1 (TGF-beta1)-responsive stem cells derived from murine bone marrow. Stringent selection of the stem cells was accomplished under low serum conditions by virtue of an inherent survival response to a TGF-beta1-vWF fusion protein that was bound to collagen matrices. The TGF-beta1-responsive stem cells initially exhibited a non-adherent and uniformly blastoid morphology, underwent expansion into colonies upon serum reconstitution, and were capable of overt cytodifferentiation along fibrogenic, osteogenic, chondrogenic, or adipogenic lineages upon growth factor stimulation. Remarkably, these stem cells also underwent rapid expansion in the presence of either hematopoietic stem cell factor (SCF) or interleukin3 (IL-3), and differentiated into myeloid and lymphoid phenotypes upon exposure to the latter. Taken together, these results support the hypothesis that pluripotent premesenchymal prehematopoietic progenitor cells, designated P4 stem cells, are present postnatally in murine bone marrow and, thus, may be summarily isolated for various cell-based experimental therapies.

Adipocytes↗
PubMed2001

Statistical model relating CA3 burst probability to recovery from burst-induced depression at recurrent collateral synapses.

When neuronal excitability is increased in area CA3 of the hippocampus in vitro, the pyramidal cells generate periodic bursts of action potentials that are synchronized across the network. We have previously provided evidence that synaptic depression at the excitatory recurrent collateral synapses in the CA3 network terminates each population burst so that the next burst cannot begin until these synapses have recovered. These findings raise the possibility that burst timing can be described in terms of the probability of recovery of this population of synapses. Here we demonstrate that when neuronal excitability is changed in the CA3 network, the mean and variance of the interburst interval change in a manner that is consistent with a timing mechanism comprised of a pool of exponentially relaxing pacemakers. The relaxation time constant of these pacemakers is the same as the time constant describing the recovery from activity-dependent depression of recurrent collateral synapses. Recovery was estimated from the rate of spontaneous transmitter release versus time elapsed since the last CA3 burst. Pharmacological and long-term alterations of synaptic strength and network excitability affected CA3 burst timing as predicted by the cumulative binomial distribution if the burst pace-maker consists of a pool of recovering recurrent synapses. These findings indicate that the recovery of a pool of synapses from burst-induced depression is a sufficient explanation for burst timing in the in vitro CA3 neuronal network. These findings also demonstrate how information regarding the nature of a pacemaker can be derived from the temporal pattern of synchronous network activity. This information could also be extracted from less accessible networks such as those generating interictal epileptiform discharges in vivo.

Algorithms↗
PubMed2000

Regulation of the meiosis-inhibited protein kinase, a p38(MAPK) isoform, during meiosis and following fertilization of seastar oocytes.

A p38(MAPK) homolog Mipk (meiosis-inhibited protein kinase) was cloned from seastar oocytes. This 40-kDa protein shares approximately 65% amino acid identity with mammalian p38-alpha isoforms. Mipk was one of the major tyrosine-phosphorylated proteins in immature oocytes arrested at the G(2)/M transition of meiosis I. The tyrosine phosphorylation of Mipk was increased in response to anisomycin, heat, and osmotic shock of oocytes. During 1-methyladenine-induced oocyte maturation, Mipk underwent tyrosine dephosphorylation and remained dephosphorylated in mature oocytes and during the early mitotic cell divisions until approximately 12 h after fertilization. At the time of differentiation and acquisition of G phases in the developing embryos, Mipk was rephosphorylated on tyrosine. In oocytes that were microinjected with Mipk antisense oligonucleotides and subsequently were allowed to mature and become fertilized, differentiation was blocked. Because MipK antisense oligonucleotides and a dominant-negative (K62R)Mipk when microinjected into immature oocytes failed to induce germinal vesicle breakdown, inhibition of Mipk function was not sufficient by itself to cause oocyte maturation. These findings point to a putative role for Mipk in cell cycle control as a G-phase-promoting factor.

Amino Acid Sequence↗
PubMed2000

Integrin-linked kinase (ILK): a "hot" therapeutic target.

Integrin-mediated cell adhesion is known to regulate gene expression through the activation of transcription factors. We have recently revealed that these activations are mediated through integrin-linked kinase (ILK). ILK is an ankyrin repeat-containing serine-threonine protein kinase that can interact directly with the cytoplasmic domain of the beta1 and beta3 integrin subunits and whose kinase activity is modulated by cell-extracellular matrix interactions. We have shown that ILK overexpression results in the translocation of beta-catenin to the nucleus, which then forms a complex formation with the lymphoid enhancer binding factor 1 (LEF-1) transcription factor, subsequently activating the transcriptional activity of promoters containing LEF-1 response elements. ILK phosphorylates the glycogen synthase kinase-3 (GSK-3), which inhibits GSK-3 activity. We have demonstrated that ILK stimulates activator protein-1 transcriptional activity through GSK-3 and the subsequent regulation of the c-Jun-DNA interaction. ILK also phosphorylates protein kinase B (PKB/Akt) and stimulates its activity. We have shown that ILK is an upstream effector of the phosphatidylinositol 3-kinase-dependent regulation of PKB/Akt. ILK has been shown to phosphorylate PKB/Akt on Ser-473 in vitro and in vivo. Our results clearly indicate that ILK is a key element in the regulation of integrin signaling as well as growth factor and Wnt signaling pathways. PTEN (phosphatase and tensin homolog detected on chromosome 10) is a tumor suppressor gene located on chromosome 10q23 that encodes a protein and phospholipid phosphatase. It is now estimated that inactivation mutants of PTEN exist in 60% of all forms of solid tumors. Loss of expression or mutational inactivation of PTEN leads to the constitutive activation of PKB/Akt via enhanced phosphorylation of Thr-308 and Ser-473. We have demonstrated that the activity of ILK is constitutively elevated in PTEN mutant cells. A small molecule ILK inhibitor suppresses the phosphorylation of PKB at the Ser-473 but not the Thr-308 site in the PTEN mutant cells. These results indicate that inhibition of ILK may be of significant value in solid tumor therapy.

Animals↗
PubMed2000

Structure-based functional classification of hypothetical protein MTH538 from Methanobacterium thermoautotrophicum.

The structure of MTH538, a previously uncharacterized hypothetical protein from Methanobacterium thermoautotrophicum, has been determined by NMR spectroscopy. MTH538 is one of numerous structural genomics targets selected in a genome-wide survey of uncharacterized sequences from this organism. MTH538 is a so-called singleton, a sequence not closely related to any other (known) sequences. The structure of MTH538 closely resembles the known structures of receiver domains from two component response regulator systems, such as CheY, and is similar to the structures of flavodoxins and GTP-binding proteins. Tests on MTH538 for characteristic activities of CheY and flavodoxin were negative. MTH538 did not become phosphorylated in the presence of acetyl phosphate and Mg(2+), although it appeared to bind Mg(2+). MTH538 also did not bind flavin mononucleotide (FMN) or coenzyme F(420). Nevertheless, sequence and structure parallels between MTH538/CheY and two families of ATPase/phosphatase proteins suggest that MTH538 may have a role in a phosphorylation-independent two-component response regulator system.

Adenosine Triphosphatases↗
PubMed2000

Solution structure of the RNA polymerase subunit RPB5 from Methanobacterium thermoautotrophicum.

RPB5 is an essential subunit of eukaryotic and archaeal RNA polymerases. It is a proposed target for transcription activator proteins in eukaryotes, but the mechanism of interaction is not known. We have determined the solution structure of the RPB5 subunit from the thermophilic archeon, Methanobacterium thermoautotrophicum. MtRBP5 contains a four-stranded beta-sheet platform supporting two alpha-helices, one on each side of the beta-sheet, resulting in an overall mushroom shape that does not appear to have any structural homologues in the structural database. The position and conservation of charged surface residues suggests possible modes of interaction with other proteins, as well as a rationale for the thermal stability of this protein.

Amino Acid Sequence↗
PubMed2000

Inhibition of rabbit keratocyte and human fetal lens epithelial cell proliferation by retrovirus-mediated transfer of antisense cyclin G1 and antisense MAT1 constructs.

The aim of this study is to evaluate the potential of gene transfer of cell cycle control genes as treatment of corneal haze or secondary cataract formation. The guiding hypothesis is that strategic modulation of the cyclin G1 or MAT1 gene by retrovirus-mediated gene transfer will inhibit proliferation of rabbit keratocytes (RabK) and fetal human lens epithelial (FHLEpi) cells in vitro. RabK and FHLEpi cell cultures were transduced in triplicate with retroviral vectors bearing either a nuclear-targeted beta-galactosidase, an antisense cyclin G1 (aG1), an antisense MAT1 (aMAT1) construct, or the neo(r) gene. The presence of beta-galactosidase activity in the transduced cultures was detected by immunohistochemical X-Gal staining, while cyclin G1 and MAT1 protein expression levels were evaluated by Western analysis. Proliferation of RabKs and FHLEpi cells was analyzed by counting the number of cells in the aG1 and aMAT1 vector-transduced cultures over 5 days. The mean transduction efficiency was 34.4% (SD 1.41) for RabKs and 19.7% (SD 1.83) for FHLEpi cells. Downregulation of cyclin G1 and MAT1 protein expression was noted 24 hr after transduction of RabK cultures with the respective vectors. Cytostatic effects of the aG1 and aMAT1 vectors in both RabKs and FHLEpi cells were most pronounced on the fifth day (RabKs, p < 0.0007; FHEpi cells, p < 0.001). An increased incidence of apoptosis was identified in both aG1 and MAT1-transduced FHLEpi cells. Taken together, these data suggest the potential utility of developing aG1 and aMAT1 retroviral vectors in gene therapy protocols for corneal haze and secondary cataract formation.

Animals↗
PubMed2000

Rapid fold and structure determination of the archaeal translation elongation factor 1beta from Methanobacterium thermoautotrophicum.

The tertiary fold of the elongation factor, aEF-1beta, from Methanobacterium thermoautotrophicum was determined in a high-throughput fashion using a minimal set of NMR experiments. NMR secondary structure prediction, deuterium exchange experiments and the analysis of chemical shift perturbations were combined to identify the protein fold as an alpha-beta sandwich typical of many RNA binding proteins including EF-G. Following resolution of the tertiary fold, a high resolution structure of aEF-1beta was determined using heteronuclear and homonuclear NMR experiments and a semi-automated NOESY assignment strategy. Analysis of the aEF-1beta structure revealed close similarity to its human analogue, eEF-1beta. In agreement with studies on EF-Ts and human EF-1beta, a functional mechanism for nucleotide exchange is proposed wherein Phe46 on an exposed loop acts as a lever to eject GDP from the associated elongation factor G-protein, aEF-1alpha. aEF-1beta was also found to bind calcium in the groove between helix alpha2 and strand beta4. This novel feature was not observed previously and may serve a structural function related to protein stability or may play a functional role in archaeal protein translation.

Archaeal Proteins↗
PubMed2000

NMR structure determination and structure-based functional characterization of conserved hypothetical protein MTH1175 from Methanobacterium thermoautotrophicum.

The solution structure of MTH1175, a 124-residue protein from the archaeon Methanobacterium thermoautotrophicum has been determined by NMR spectroscopy. MTH1175 is part of a family of conserved hypothetical proteins (COG1433) with unknown functions which contains multiple paralogs from all complete archaeal genomes and the archaeal gene-rich bacterium Thermotoga maritima. Sequence similarity indicates this protein family may be related to the nitrogen fixation proteins NifB and NifX. MTH1175 adopts an alpha/beta topology with a single mixed beta-sheet, and contains two flexible loops and an unstructured C-terminal tail. The fold resembles that of Ribonuclease H and similar proteins, but differs from these in several respects, and is not likely to have a nuclease activity.

Amino Acid Sequence↗
PubMed2000

Structural proteomics of an archaeon.

A set of 424 nonmembrane proteins from Methanobacterium thermoautotrophicum were cloned, expressed and purified for structural studies. Of these, approximately 20% were found to be suitable candidates for X-ray crystallographic or NMR spectroscopic analysis without further optimization of conditions, providing an estimate of the number of the most accessible structural targets in the proteome. A retrospective analysis of the experimental behavior of these proteins suggested some simple relations between sequence and solubility, implying that data bases of protein properties will be useful in optimizing high throughput strategies. Of the first 10 structures determined, several provided clues to biochemical functions that were not detectable from sequence analysis, and in many cases these putative functions could be readily confirmed by biochemical methods. This demonstrates that structural proteomics is feasible and can play a central role in functional genomics.

Cloning, Molecular↗
PubMed2000

Characterization of differential gene expression in monkey arterial neointima following balloon catheter injury.

Vaso-occlusive sequelae following percutaneous transluminal coronary angioplasty (PTCA), including smooth muscle cell migration, proliferation, and attendant extracellular matrix production, often results in restenosis of the treated artery. To further understand the molecular mechanisms governing progressive intimal hyperplasia, we performed a molecular screen using differential display PCR on total RNA prepared from injured and normal carotid arterial segments to identify a subset of differentially expressed genes at t=7 days post-balloon catheter injury in a non-human primate. DNA sequence analysis of selected differentially expressed RNA by this procedure using 240 combinations of random primer pairs yielded 41 distinct cDNA sequences: 22 of which have significant sequence homology to previously identified meta-zoan genes, 15 GEMS (genes expressed in monkey neointima), and 4 GSMS (genes suppressed in monkey neointima) that have little homology to reported sequences. Among the up-regulated homologues include i) secreted growth regulatory factors, ii) membrane receptors, iii) transcription factors, iv) cell adhesion molecules, and v) extracellular matrix proteins; some of which have not been previously linked to vascular restenosis. In particular, Cyr61, a known angiogenesis inducer, was found to be highly expressed in the neointima lesion of the balloon-injured monkey artery. This finding provides the first links of Cyr61 to the pathogenesis of vascular restenosis, and identifies a novel locus for potential therapeutic intervention. These studies identified a number of known and unknown genes, whose up- or down-regulated expression during the proliferative phase of vascular restenosis makes them potential targets for therapeutic intervention.

Animals↗
PubMed1999

Characterization of fertilization-modulated myelin basic protein kinases from sea star: regulation of Mapk.

The myelin basic protein (MBP)-phosphorylating enzymes present during maturation and early embryogenesis of the sea star (Pisaster ochraceus) were investigated. The major maturation-activated MBP kinase (p45 Mapk) was molecularly cloned based on tryptic sequence information obtained with the purified enzyme and shown to be highly related to human Erk1 with 76% amino acid identity. Kinase assays and immunoblotting studies revealed that Mapk remained highly active until 12 h post-fertilization (PF), after which it declined. By 4 days PF, Mapk protein was no longer detectable. At 3 h PF, about half of the detectable MBP phosphotransferase activity could be attributed to a 75 kDa protein kinase that was distinct from Mapk. Like Mapk, this protein phosphorylated MBP mostly on threonine residues, but it failed to phosphorylate a peptide (APRTPGGRR) based upon the Thr-97 MAP kinase phosphorylation site in MBP. Rather, it phosphorylated a peptide (AAQKRPSQRTKYLA) patterned after the N-terminus of MBP. Our studies also showed a dramatic increase in MBP phosphotransferase activity occurred by 4 days PF that arose from a third kinase that phosphorylated MBP solely on serine residues. This kinase exhibited the following substrate substrate preference: AAQKRPSQRTKYLA, peptide substrate for S6 kinases (AKRRRLSSLRASTSKSESSQK) > MBP > histone H1 > prota-mine > casein > APRTPGGRR. This kinase was not appreciably affected by addition of phosphatidylserine/diacylglycerol, or the staurosporine analogue Roche Compound 3, but it was partly inhibited by a protein kinase C pseudosubstrate peptide. Gel filtration analysis revealed an apparent molecular mass of 41 kDa for the enzyme. Therefore, at least two novel MBP-phosphorylating enzymes distinct from Mapk are preferentially activated following fertilization and early embryogenesis of the sea star.

Amino Acid Sequence↗
PubMed1999

Backbone dynamics of detergent-solubilized alamethicin from amide hydrogen exchange measurements.

Alamethicin is a 20 amino acid antibiotic peptide produced by the soil fungus Trichoderma viride. The peptide inserts into bacterial membranes and self-associates to form ion channels, but the details of this process are unknown. Residue-specific acid- and base-catalyzed exchange data were obtained for 16 of 18 backbone amides of alamethicin dissolved in sodium dodecyl sulfate micelles using high-resolution 2-dimensional heteronuclear nuclear magnetic resonance spectroscopy. To facilitate interpretation of the exchange data, we synthesized N-acetyl-alpha-aminoisobutyric acid-N'-methyl and N-acetyl-alanine-N'-methyl and measured the pD dependence of their hydrogen-deuterium exchange rates to determine the sequence-dependent inductive and steric effects of the alpha-aminoisobutyric acid residue. Intramolecular H-bonding in alamethicin was monitored through the exchange parameters kmin (minimum exchange rate) which indicate that the backbone is significantly more stable than the backbones of alanine-based helical peptides. Rapid exchange at Gly-11 suggests a highly local conformational flexibility in the middle of the peptide. Interactions with the detergent micelle were revealed by the exchange parameters pDmin (pD of minimum exchange) which suggest that the N-terminus of alamethicin interacts more strongly with the detergent micelle than does the C-terminus. A periodicity in pDmin difference data reveals that one surface of the helix interacts more strongly with the micelle. The surface consists of residues 1, 5, 9, 13, 16, and 20. The opposite face of the helix contains several polar residues (two glutamines and a glycine), suggesting that, on average, this face of the helix is directed toward the solvent. These results serve as a model for the interaction of the peptide with membranes containing anionic lipid. In combination with published molecular dynamics simulations [Gibbs et al. (1997) Biophys. J. 72, 2490-2495], the present results also offer insight into the mechanisms of hydrogen-deuterium exchange in helical peptides.

Alamethicin↗