The t(8;21) translocation, found in adult acute myelogenous leukemia, results in the formation of an AML1/ETO chimeric transcription factor. AML1/ETO expression leads to alterations in hematopoietic progenitor cell differentiation, although its role in leukemic transformation is not clear. The N-terminal portion of AML1, which is retained in AML1/ETO, contains a region of homology to the FAST proteins, which cooperate with Smads to regulate transforming growth factor beta1 (TGF-beta1) target genes. We have demonstrated the physical association of Smad proteins with AML1 and AML1/ETO by immunoprecipitation and have mapped the region of interaction to the runt homology domain in these AML1 proteins. Using confocal microscopy, we demonstrated that AML1, and ETO and/or AML1/ETO, colocalize with Smads in the nucleus of t(8;21)-positive Kasumi-1 cells, in the presence but not the absence of TGF-beta1. Using transient transfection assays and a reporter gene construct that contains both Smad and AML1 consensus binding sequences, we demonstrated that overexpression of AML1B cooperates with TGF-beta1 in stimulating reporter gene activity, whereas AML1/ETO represses basal promoter activity and blocks the response to TGF-beta1. Considering the critical role of TGF-beta1 in the growth and differentiation of hematopoietic cells, interference with TGF-beta1 signaling by AML1/ETO may contribute to leukemogenesis.

3T3 Cells↗

PubMed2000

Highly specific, membrane-permeant peptide blockers of cGMP-dependent protein kinase Ialpha inhibit NO-induced cerebral dilation.

Arrays of octameric peptide libraries on cellulose paper were screened by using (32)P-autophosphorylated cGMP-dependent protein kinase Ialpha (cGPK) to identify peptide sequences with high binding affinity for cGPK. Iterative deconvolution of every amino acid position in the peptides identified the sequence LRK(5)H (W45) as having the highest binding affinity. Binding of W45 to cGPK resulted in selective inhibition of the kinase with K(i) values of 0.8 microM and 560 microM for cGPK and cAMP-dependent protein kinase (cAPK), respectively. Fusion of W45 to membrane translocation signals from HIV-1 tat protein (YGRKKRRQRRRPP-LRK(5)H, DT-2) or Drosophila Antennapedia homeo-domain (RQIKIWFQNRRMKWKK-LRK(5)H, DT-3) proved to be an efficient method for intracellular delivery of these highly charged peptides. Rapid translocation of the peptides into intact cerebral arteries was demonstrated by using fluorescein-labeled DT-2 and DT-3. The inhibitory potency of the fusion peptides was even greater than that for W45, with K(i) values of 12.5 nM and 25 nM for DT-2 and DT-3, respectively. Both peptides were still poor inhibitors of cAPK. Selective inhibition of cGPK by DT-2 or DT-3 in the presence of cAPK was demonstrated in vitro. In pressurized cerebral arteries, DT-2 and DT-3 substantially decreased NO-induced dilation. This study provides functional characterization of a class of selective cGPK inhibitor peptides in vascular smooth muscle and reveals a central role for cGPK in the modulation of vascular contractility.

Animals↗

PubMed2000

[Polymorphic ventricular tachycardia after intravenous adenosine].

Explore the source record for details and available documents.

Adenosine↗

PubMed2000

The amyloid precursor-like protein 2 associates with the major histocompatibility complex class I molecule K(d).

Amyloid precursor-like protein 2 (APLP2) is a member of a protein family related to the amyloid precursor protein, which is implicated in Alzheimer's disease. Little is known about the physiological function of this protein family. The adenovirus E3/19K protein binds to major histocompatibility complex (MHC) class I antigens in the endoplasmic reticulum, thereby preventing their transport to the cell surface. In cells coexpressing E3/19K and the MHC K(d) molecule, K(d) is associated with E3/19K and two cellular protein species with masses of 100 and 110 kDa, termed p100/110. Interestingly, p100/110 are released from the complex upon the addition of K(d)-binding peptides, suggesting a role for these proteins in peptide transfer to MHC molecules. Here we demonstrate by microsequencing, reactivity with APLP2-specific antibodies, and comparison of biochemical parameters that p100/110 is identical to human APLP2. We further show that the APLP2/K(d) association does not require the physical presence of E3/19K. Thus, APLP2 exhibits an intrinsic affinity for the MHC K(d) molecule. Similar to the binding of MHC molecules to the transporter associated with antigen processing, complex formation between APLP2 and K(d) strictly depends upon the presence of beta(2)-microglobulin. Conditions that prolong the residency of K(d) in the endoplasmic reticulum lead to a profound increase of the association and a drastic reduction of APLP2 transport. Therefore, this unexpected interplay between these unrelated molecules may have implications for both MHC antigen and APLP2 function.

Amino Acid Sequence↗

PubMed2000

Specificity determinants of substrate recognition by the protein kinase DYRK1A.

DYRK1A is a dual-specificity protein kinase that is thought to be involved in brain development. We identified a single phosphorylated amino acid residue in the DYRK substrate histone H3 (threonine 45) by mass spectrometry, phosphoamino acid analysis, and protein sequencing. Exchange of threonine 45 for alanine abolished phosphorylation of histone H3 by DYRK1A and by the related kinases DYRK1B, DYRK2, and DYRK3 but not by CLK3. In order to define the consensus sequence for the substrate specificity of DYRK1A, a library of 300 peptides was designed in variation of the H3 phosphorylation site. Evaluation of the phosphate incorporation into these peptides identified DYRK1A as a proline-directed kinase with a phosphorylation consensus sequence (RPX(S/T)P) similar to that of ERK2 (PX(S/T)P). A peptide designed after the optimal substrate sequence (DYRKtide) was efficiently phosphorylated by DYRK1A (K(m) = 35 microM) but not by ERK2. Both ERK2 and DYRK1A phosphorylated myelin basic protein, whereas only ERK2, but not DYRK1A, phosphorylated the mitogen-activated protein kinase substrate ELK-1. This marked difference in substrate specificity between DYRK1A and ERK2 can be explained by the requirement for an arginine at the P -3 site of DYRK substrates and its presumed interaction with aspartate 247 conserved in all DYRKs.

Amino Acid Sequence↗

PubMed2000

Enrichment of hydrophobic proteins via Triton X-114 phase partitioning and hydroxyapatite column chromatography for mass spectrometry.

Membrane proteins are the starting point of several signal transduction pathways. Therefore, the separation and identification of these proteins are of great interest in proteome analysis. However, the specific properties of membrane proteins seriously impede their analysis. We present an effective and highly reproducible method for the two-dimensional separation of extremely hydrophobic proteins and demonstrate the advantages of special preseparation procedures for the identification of proteins which have very similar Mr and p/. Using the example of the integral membrane protein very low density lipoprotein (VLDL) receptor (NCBI Acc. # 1730111) and the soluble heat shock protein (HSP) 90 (NCBI Acc. # 386786) we present the applicability of a phase-separation system with Triton X-114. Using matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) of the protein spots after 2-D separation of the hydrophilic and the strongly hydrophobic protein fraction of human endothelial cells (ECV cell line), we were able to distinguish both proteins.

Cell Line↗

PubMed2000

Towards a two-dimensional proteome map of Mycoplasma pneumoniae.

A Proteome map of the bacterium Mycoplasma pneumoniae was constructed using two-dimensional (2-D) gel electrophoresis in combination with mass spectrometry (MS). M. pneumoniae is a human pathogen with a known genome sequence of 816 kbp coding for only 688 open reading frames, and is therefore an ideal model system to explore the scope and limits of the current technology. The soluble protein content of this bacterium grown under standard laboratory conditions was separated by 1-D or 2-D gel electrophoresis applying various pH gradients, different acrylamide concentrations and buffer systems. Proteins were identified using liquid chromatography-electrospray ionization ion trap and matrix-assisted laser desorption/ionization-MS. Mass spectrometric protein identification was supported and controlled using N-terminal sequencing and immunological methods. So far, proteins from about 350 spots were characterized with MS by determining the molecular weights and partial sequences of their tryptic peptides. Comparing these experimental data with the DNA sequence-derived predictions it was possible to assign these 350 proteins to 224 genes. The importance of proteomics for genome analysis was shown by the identification of four proteins, not annotated in the original publication. Although the proteome map is still incomplete, it is already a useful reference for comparative analyses of M. pneumoniae cells grown under modified conditions.

Acrylic Resins↗

PubMed2000

Yield of renal arteriography in the evaluation of pediatric hypertension.

The prevalence of renovascular disease is estimated to be 3%-5% in pediatric patients with hypertension. The utility of non-invasive imaging tests has not been evaluated in children, and renal arteriography remains the diagnostic test of choice. However, there are no established guidelines for the application of this test and information is not available about the likelihood of detecting an abnormality if an arteriogram is performed in children with hypertension. Therefore, we reviewed the yield of renal arteriography in pediatric patients if the test was performed based on the following two criteria: (1) severe hypertension exceeding the 99th percentile for age and sex or (2) failure to control high blood pressure with one antihypertensive drug. During the period 1983-1998, 28 children (mean age 11.7 years) who satisfied one of the above criteria underwent renal arteriography to investigate hypertension. None of the patients were renal transplant recipients. The average duration of hypertension was 11 months and the peak blood pressure was 168/107 mmHg. The renal arteriogram was abnormal in 12 patients (43%). Unilateral renal artery stenosis was the most-common abnormality. When the patients were divided into two groups - those with an abnormal or normal test result - they did not differ in age, sex, or racial distribution. The peak systolic blood pressure was higher in children with an abnormal renal arteriogram (P<0.05). Among those undergoing the arteriogram on the basis of the first criterion, i.e., severe hypertension, 11 of 23 (48%) studies were abnormal. Five children had an arteriogram based on the second criterion--failure to control the blood pressure with one medication--and in 1 patient (20%) the test was abnormal. We conclude that the prevalence of renovascular disease in a population of hypertensive children subjected to renal arteriography is around 40%. Two clinical criteria--namely severe hypertension or failure to control hypertension effectively with one drug--are useful to guide the application of renal arteriography in children with hypertension.

Adolescent↗

PubMed2000

Bartter syndrome and focal segmental glomerulosclerosis: a possible link between two diseases.

We describe a patient with signs and symptoms of classic Bartter syndrome. The patient tested negative for all known genetic abnormalities associated with this tubular disorder. Proteinuria was found within 1 year after the diagnosis of Bartter syndrome. A renal biopsy performed 6 months later, when her kidney function was normal, revealed focal segmental glomerulosclerosis (FSGS). We propose a link between stimulation of the renin-angiotensin system and sclerotic changes in the glomerulus. This lesion may explain previous reports of kidney failure in patients with Bartter syndrome.

Adolescent↗

PubMed2000

Ventricular tachycardia catheter ablation in arrhythmogenic right ventricular dysplasia: a 16-year experience.

Arrhythmogenic right ventricular dysplasia (ARVD) is a structural heart disease affecting young adults that leads to cardiac rhythm disorders including supraventricular and mostly ventricular arrhythmias. Sudden death may be the first presentation of the disease. Ablation techniques have been used for the treatment of ventricular tachycardia in cases resistant to drug therapy. Radiofrequency is appropriate as a first approach for ventricular tachycardia ablation in ARVD; however, its effectiveness is less than 40% at the first session. Fulguration is effective for ventricular tachy-cardia ablation and should be used in the same session after ineffective radiofrequency ablation. However, fulguration requires expertise, general anesthesia, and more than one session in half of all patients. Radiofrequency and fulguration plus other common forms of treatment including pacemakers and automatic implantable cardioverter defibrillators provides a clinical success rate of 81% to 93% in a series of 50 consecutive patients studied during 16 years. Earlier poor reputation of fulguration was the result of poorly understood technical problems concerning the physics and biophysics of the procedure under control with presently available methods. This in-depth study of a large population over a long time period demonstrates that fulguration should be rehabilitated.

Adolescent↗

PubMed2000

Food neophobia in childhood affects dietary variety.

OBJECTIVE: To determine whether children with food neophobia (unwillingness to try new foods) have more restrictive diets than children without neophobia. SUBJECTS: Seventy children were classified into 3 groups based on scores obtained on the Food Neophobia Scale: neophobic group, score greater than 41; neophilic group, score less than 27; and average group, score of 28 to 40. DESIGN: Dietary data were collected and analyzed for 3 days selected randomly. The dependent variables measured were energy and nutrient intakes, servings of each Food Guide Pyramid group, and Health Eating Index (HEI) scores. STATISTICAL ANALYSES: chi 2, 1-way analysis of covariance, and Scheffé multiple comparisons tests were conducted. RESULTS: The 3 groups were similar with respect to the number of children meeting two thirds of the RDA/DRI for energy and most nutrients. The exception was vitamin E: fewer neophobic children met two thirds of the recommended value for this nutrient than average and neophilic children. The overall HEI score was significantly lower for the neophobic group compared with the average and neophilic groups. The HEI index showed that children with neophobia had a higher intake of saturated fat and less food variety than children without food neophobia. APPLICATIONS: Dietitians should emphasize increased food variety for children within the context of a healthful diet. Research should be conducted to determine the effects of dietary variety on quality of diet and health of children.

Black or African American↗

PubMed2000

Tubulin-tyrosine ligase, a long-lasting enigma.

Tubulins and microtubules are subjected to several post-translational modifications of which the reversible detyrosination/tyrosination of the carboxy-terminal end of most alpha-tubulins has been extensively analysed. This modification cycle involves a specific carboxypeptidase and the activity of the tubulin-tyrosine ligase (TTL). The true physiological function of TTL has so far not been established. This review describes the purification of TTL to homogeneity by biochemical methods, its in vitro properties and the generation of monoclonal antibodies. These mabs not only enabled a very convenient and rapid purification of TTL by immunoaffinity chromatography but also its extensive characterization by protein sequencing, which led to the isolation of the full length cDNA. With this information, gene disruption should be feasible in order to determine the physiological significance of the tyrosination cycle.

Amino Acid Sequence↗

PubMed2000

Inhaled particle-bound sulfate: effects on pulmonary inflammatory responses and alveolar macrophage function.

Acid sulfate-coated solid particles are a significant environmental hazard produced primarily by the combustion of fossil fuels. We have previously described a system for the nascent generation of carbonaceous particles surface coated with approximately 140 microg/m(3) acid sulfate [cpSO(4)(2-); 10 mg/m(3) carbon black (CB) and 10 ppm sulfur dioxide (SO(2)) at 85% relative humidity (RH)]. The effects of inhaled cpSO(4)(2-) on pulmonary host defenses are assessed in the present work. Mice were acutely exposed (4 h) to either 10 mg/m(3) CB, 10 ppm SO(2), or their combination at 10% or 85% RH in a nose-only inhalation chamber. No evidence of an inflammatory response was found following any of the exposures as assessed by total cell counts and differential cell counts from bronchoalveolar lavage fluid. However, alveolar macrophage Fc receptor-mediated phagocytosis decreased only following exposure to 140 microg cpSO(4)(2-), significant suppression occurred after 24 h, maximal suppression occurred at 3 days postexposure, and recovery to preexposure levels required 7-14 days. Intrapulmonary bactericidal activity (IBA) was also suppressed only after exposure to 140 microg cpSO(4)(2-); suppression was maximal at 1 day postexposure and recovered by day 7. To assess the effects of lower cpSO(4)(2-) concentrations, mice were repeatedly exposed to 1 mg/m(3) CB and 1 ppm SO(2) at 85% RH ( approximately 20 microg/m(3) cpSO(4)(2-) for 4 h/day) for up to 6 days. A significant decrement in IBA was observed following 5 and 6 days of exposure. These studies indicated that acute or repeated exposure to cpSO(4)(2-) could alter pulmonary host defense mechanisms.

Administration, Inhalation↗