BRK phosphorylates SMAD4 for proteasomal degradation and inhibits tumor suppressor FRK to control SNAIL, SLUG, and metastatic potential.

The tumor-suppressing function of SMAD4 is frequently subverted during mammary tumorigenesis, leading to cancer growth, invasion, and metastasis. A long-standing concept is that SMAD4 is not regulated by phosphorylation but ubiquitination. Our search for signaling pathways regulated by breast tumor kinase (BRK), a nonreceptor protein tyrosine kinase that is up-regulated in ~80% of invasive ductal breast tumors, led us to find that BRK competitively binds and phosphorylates SMAD4 and regulates transforming growth factor-ß/SMAD4 signaling pathway. A constitutively active BRK (BRK-Y447F) phosphorylates SMAD4, resulting in its recognition by the ubiquitin-proteasome system, which accelerates SMAD4 degradation. Activated BRK-mediated degradation of SMAD4 is associated with the repression of tumor suppressor gene FRK and increased expression of mesenchymal markers, SNAIL, and SLUG. Thus, our data suggest that combination therapies targeting activated BRK signaling may have synergized the benefits in the treatment of SMAD4 repressed cancers.

Epidemiologic factors and urogenital infections associated with preterm birth in a midwestern U.S. population.

OBJECTIVE: To correlate epidemiologic factors with urogenital infections associated with preterm birth. METHODS: Pregnant women were sequentially included from four Wisconsin cohorts: large urban, midsize urban, small city, and rural city. Demographic, clinical, and current pregnancy data were collected. Cervical and urine specimens were analyzed by microscopy, culture, and polymerase chain reaction for potential pathogens. RESULTS: Six hundred seventy-six women were evaluated. Fifty-four (8.0%) had preterm birth: 12.1% (19/157) large urban, 8.8% (15/170) midsize urban, 9.4% (16/171) small city, and 2.3% (4/178) rural city. Associated host factors and infections varied significantly among sites. Urogenital infection rates, especially Mycoplasma hominis and Ureaplasma parvum, were highest at the large urban site. Large urban site, minority ethnicity, multiple infections, and certain historical factors were associated with preterm birth by univariable analysis. By multivariable analysis, preterm birth was associated with prior preterm birth (adjusted odds ratio [aOR] 2.76, 95% confidence interval [CI] 1.27-6.02) and urinary tract infection (aOR 2.62, 95% CI 1.32-519), and negatively associated with provider-assessed good health (aOR 0.42, 95% CI 0.23-0.76) and group B streptococcal infection treatment (surrogate for health care use) (aOR 0.38, 95% CI 0.15-.99). Risk and protective factors were similar for women with birth at less than 35 weeks, and additionally associated with M hominis (aOR 3.6, 95% CI 1.4-9.7). CONCLUSION: These measured differences among sites are consistent with observations that link epidemiologic factors, both environmental and genetic, with minimally pathogenic vaginal bacteria, inducing preterm birth, especially at less than 35 weeks of gestation.

Proteomic analysis of chromatin-modifying complexes in Saccharomyces cerevisiae identifies novel subunits.

Epigenetics is the alteration of phenotype without affecting the genotype. An underlying molecular mechanism of epigenetics is the changes of chromatin structure by covalent histone modifications and nucleosome reorganization. In the yeast, Saccharomyces cerevisiae, two of the most well-studied macromolecular complexes that perform these epigenetic changes are the ATP-dependent Swi/Snf chromatin-remodelling complex and the SAGA histone acetyltransferase complex. To understand fully the mechanism by which these large protein complexes perform their functions in the cell, it is crucial that all the subunits of these complexes are identified. In an attempt to identify new subunits associated with SAGA and Swi/Snf, we used tandem affinity purification, followed by a multidimensional protein identification technology to analyse the subunit composition. Our analysis identified two novel proteins, one associated with SAGA, YPL047W (Sgf11), and another associated with Swi/Snf, Rtt102.

Large-scale analysis of the yeast proteome by multidimensional protein identification technology.

We describe a largely unbiased method for rapid and large-scale proteome analysis by multidimensional liquid chromatography, tandem mass spectrometry, and database searching by the SEQUEST algorithm, named multidimensional protein identification technology (MudPIT). MudPIT was applied to the proteome of the Saccharomyces cerevisiae strain BJ5460 grown to mid-log phase and yielded the largest proteome analysis to date. A total of 1,484 proteins were detected and identified. Categorization of these hits demonstrated the ability of this technology to detect and identify proteins rarely seen in proteome analysis, including low-abundance proteins like transcription factors and protein kinases. Furthermore, we identified 131 proteins with three or more predicted transmembrane domains, which allowed us to map the soluble domains of many of the integral membrane proteins. MudPIT is useful for proteome analysis and may be specifically applied to integral membrane proteins to obtain detailed biochemical information on this unwieldy class of proteins.

Polyhydroxybenzoates inhibit ascorbic acid activation of mitochondrial glycerol-3-phosphate dehydrogenase: implications for glucose metabolism and insulin secretion.

Glycerol-3-phosphate dehydrogenase from pig brain mitochondria was stimulated 2.2-fold by the addition of 50 microm l-ascorbic acid. Enzyme activity, dependent upon the presence of l-ascorbic acid, was inhibited by lauryl gallate, propyl gallate, protocatechuic acid ethyl ester, and salicylhydroxamic acid. Homogeneous pig brain mitochondrial glycerol-3-phosphate dehydrogenase was activated by either 150 microm L-ascorbic acid (56%) or 300 microm iron (Fe(2+) or Fe(3+) (62%)) and 2.6-fold by the addition of both L-ascorbic acid and iron. The addition of L-ascorbic acid and iron resulted in a significant increase of k(cat) from 21.1 to 64.1 s(-1), without significantly increasing the K(m) of L-glycerol-3-phosphate (10.0-14.5 mm). The activation of pure glycerol-3-phosphate dehydrogenase by either L-ascorbic acid or iron or its combination could be totally inhibited by 200 microm propyl gallate. The metabolism of [5-(3)H]glucose and the glucose-stimulated insulin secretion from rat insulinoma cells, INS-1, were effectively inhibited by 500 microm or 1 mm propyl gallate and to a lesser extent by 5 mm aminooxyacetate, a potent malate-aspartate shuttle inhibitor. The combined data support the conclusion that l-ascorbic acid is a physiological activator of mitochondrial glycerol-3-phosphate dehydrogenase, that the enzyme is potently inhibited by agents that specifically inhibit certain classes of di-iron metalloenzymes, and that the enzyme is chiefly responsible for the proximal signal events in INS-1 cell glucose-stimulated insulin release.

An automated multidimensional protein identification technology for shotgun proteomics.

We describe an automated method for shotgun proteomics named multidimensional protein identification technology (MudPIT), which combines multidimensional liquid chromatography with electrospray ionization tandem mass spectrometry. The multidimensional liquid chromatography method integrates a strong cation-exchange (SCX) resin and reversed-phase resin in a biphasic column. We detail the improvements over a system described by Link et al. (Link, A. J.; Eng, J.; Schieltz, D. M.; Carmack, E.; Mize, G. J.; Morris, D. R.; Garvik, B. M.; Yates, J. R., III. Nat. Biotechnol. 1999, 17, 676-682) that separates and acquires tandem mass spectra for thousands of peptides. Peptides elute off the SCX phase by increasing pI, and elution off the SCX material is evenly distributed across an analysis. In addition, we describe the chromatographic benchmarks of MudPIT. MudPIT was reproducible within 0.5% between two analyses. Furthermore, a dynamic range of 10000 to 1 between the most abundant and least abundant proteins/peptides in a complex peptide mixture has been demonstrated. By improving sample preparation along with separations, the method improves the overall analysis of proteomes by identifying proteins of all functional and physical classes.

Facilitating critical thinking.

Supporting staff to think effectively is essential to improve clinical systems, decrease errors and sentinel events, and engage staff involvement to refine patient care systems in readiness for new care-delivery models that truly reflect the valued role of the RN. The authors explore practical methods, based on current research and national consulting experience, to facilitate the development of mature critical thinking skills. Assessment tools, a sample agenda for formal presentations, and teaching strategies using behavioral examples that make the important and necessary link of theory to reality are discussed in the form of a critical thinking test as well as a conceptual model for application in problem solving.

Analysis of the microbial proteome.

Proteomics has begun to provide insight into the biology of microorganisms. The combination of proteomics with genetics, molecular biology, protein biochemistry and biophysics is particularly powerful, resulting in novel methods to analyse complex protein mixtures. Emerging proteomic technologies promise to increase the throughput of protein identifications from complex mixtures and allow for the quantification of protein expression levels.

Recombinant human receptors and functional assays in the discovery of altinicline (SIB-1508Y), a novel acetylcholine-gated ion channel (nAChR) agonist.

Neuronal nicotinic acetylcholine receptors (nAChRs) are a class of ion channels with significant potential as molecular targets for the design of drugs to treat a variety of CNS disorders. The discovery that neuronal nAChRs are further subdivided into multiple subtypes suggests that drugs which act selectively at specific nAChR subtypes might effectively treat Parkinson's disease (PD), Alzheimer's disease (AD), schizophrenia, ADHD, depression, anxiety or pain without the accompanying adverse side effects associated with non-selective agents such as nicotine (1) and epibatidine. Altinicline (SIB-1508Y) is a novel, small molecule designed to selectively activate neuronal nAChRs and is undergoing clinical evaluation for the treatment of PD. It was selected from a series of compounds primarily on the basis of results from functional assays, including (a) measurement of Ca2+ flux in stable cell lines expressing specific recombinant human neuronal nAChR subtypes; (b) determination of in vitro and in vivo neurotransmitter release; (c) in vivo models of PD. Biological data on both altinicline and the series of compounds from which it was selected are reported.

Intuition in professional practice. Executive and staff perceptions.

Intuition, as part of our thinking, is often misunderstood or held in mystical regard. It is important to understand the aspects of this instinctual process and to appreciate the differing perspectives of both the registered nurse in the staff position and the executive in a leadership role. We compare responses to a Socratic dialogue of the definition of intuition and offer suggestions for nursing leaders to develop clinical judgment using nursing examples of intuition.

UB-165: a novel nicotinic agonist with subtype selectivity implicates the alpha4beta2* subtype in the modulation of dopamine release from rat striatal synaptosomes.

Presynaptic nicotinic acetylcholine receptors (nAChRs) on striatal synaptosomes stimulate dopamine release. Partial inhibition by the alpha3beta2-selective alpha-conotoxin-MII indicates heterogeneity of presynaptic nAChRs on dopamine terminals. We have used this alpha-conotoxin and UB-165, a novel hybrid of epibatidine and anatoxin-a, to address the hypothesis that the alpha-conotoxin-MII-insensitive subtype is composed of alpha4 and beta2 subunits. UB-165 shows intermediate potency, compared with the parent molecules, at alpha4beta2* and alpha3-containing binding sites, and resembles epibatidine in its high discrimination of these sites over alpha7-type and muscle binding sites. (+/-)-Epibatidine, (+/-)-anatoxin-a, and (+/-)-UB-165 stimulated [(3)H]-dopamine release from striatal synaptosomes with EC(50) values of 2.4, 134, and 88 nM, and relative efficacies of 1:0.4:0.2, respectively. alpha-Conotoxin-MII inhibited release evoked by these agonists by 48, 56, and 88%, respectively, suggesting that (+/-)-UB-165 is a very poor agonist at the alpha-conotoxin-MII-insensitive nAChR subtype. In assays of (86)Rb(+) efflux from thalamic synaptosomes, a model of an alpha4beta2* nAChR response, (+/-)-UB-165 was a very weak partial agonist; the low efficacy of (+/-)-UB-165 at alpha4beta2 nAChR was confirmed in Xenopus oocytes expressing various combinations of human nAChR subunits. In contrast, (+/-)-UB-165 and (+/-)-anatoxin-a were similarly efficacious and similarly sensitive to alpha-conotoxin-MII in increasing intracellular Ca(2+) in SH-SY5Y cells, a functional assay for native alpha3-containing nAChR. These data support the involvement of alpha4beta2* nAChR in the presynaptic modulation of striatal dopamine release and illustrate the utility of exploiting a novel partial agonist, together with a selective antagonist, to dissect the functional roles of nAChR subtypes in the brain.

Seven steps to shift from tasks to outcomes.

Seven managerial strategies guide the evolution of nurses' abilities to plan patient outcomes rather than focus on tasks. The authors emphasize revitalizing report rituals to include discussion of patient-focused short- and long-term outcomes.

Individual and organizational accountability for development of critical thinking.

Advanced abilities to think critically are necessary to improve clinical systems, decrease errors and sentinel events, and engage staff involvement to refine patient care systems. The authors explore practical methods, based on current research, to promote mature critical-thinking skills in healthcare professionals, from patient care clinical judgment to a leadership perspective. Individual and organizational accountabilities for expanding critical thinking skills are investigated, and a problem-solving model is proposed as a coordinated strategy.

Identification of the dehydroascorbic acid reductase and thioltransferase (Glutaredoxin) activities of bovine erythrocyte glutathione peroxidase.

Bovine erythrocyte glutathione (GSH) peroxidase (GPX, EC 1.11.1.9) was examined for GSH-dependent dehydroascorbate (DHA) reductase (EC 1.8.5.1) and thioltransferase (EC 1.8.4.1) activities. Using the direct assay method for GSH-dependent DHA reductase activity, GPX had a kcat (app) of 140 +/- 9 min-1 and specificity constants (kcat/Km(app)) of 5.74 +/- 0.78 x 10(2) M-1s-1 for DHA and 1.18 +/- 0.17 x 10(3) M-1s-1 for GSH based on the monomer Mr of 22,612. Using the coupled assay method for thioltransferase activity, GPX had a kcat (app) of 186 +/- 9 min-1 and specificity constants (app) of 1. 49 +/- 0.14 x 10(3) M-1s-1 for S-sulfocysteine and 1.51 +/- 0.18 x 10(3) M-1s-1 for GSH based on the GPX monomer molecular weight. GPX has a higher specificity constant for S-sulfocysteine than DHA, and both assay systems gave nearly identical specificity constants for GSH. The DHA reductase and thioltransferase activities of GPX adds to the repertoire of functions of this enzyme as an important protector against cellular oxidative stress.

Structure and functional characterization of a novel human low-voltage activated calcium channel.

We have isolated and characterized overlapping cDNAs encoding a novel, voltage-gated Ca2+ channel alpha1 subunit, alpha1H, from a human medullary thyroid carcinoma cell line. The alpha1H subunit is structurally similar to previously described alpha1 subunits. Northern blot analysis indicates that alpha1H mRNA is expressed throughout the brain, primarily in the amygdala, caudate nucleus, and putamen, as well as in several nonneuronal tissues, with relatively high levels in the liver, kidney, and heart. Ba2+ currents recorded from human embryonic kidney 293 cells transiently expressing alpha1H activated at relatively hyperpolarized potentials (-50 mV), rapidly inactivated (tau = 17 ms), and slowly deactivated. Similar results were observed in Xenopus oocytes expressing alpha1H. Single-channel measurements in human embryonic kidney 293 cells revealed a single-channel conductance of approximately 9 pS. These channels are blocked by Ni2+ (IC50 = 6.6 microM) and the T-type channel antagonists mibefradil (approximately 50% block at 1 microM) and amiloride (IC50 = 167 microM). Thus, alpha1H-containing channels exhibit biophysical and pharmacological properties characteristic of low voltage-activated, or T-type, Ca2+ channels.

The catalytic mechanism of the glutathione-dependent dehydroascorbate reductase activity of thioltransferase (glutaredoxin).

The catalytic mechanism of the glutathione (GSH)-dependent dehydroascorbic acid (DHA) reductase activity of recombinant pig liver thioltransferase (RPLTT) was investigated. RPLTT and the C25S mutant protein had equivalent specificity constants (kcat/Km) for both DHA and GSH. Iodoacetamide (IAM) inactivated the DHA reductase activities of RPLTT and C25S, confirming the essential role of cysteine in the reaction mechanism. When preincubated with DHA, RPLTT but not C25S was protected against IAM inactivation, suggesting that RPLTT has the ability to chemically reduce DHA forming ascorbic acid (AA) and the intramolecular disulfide form of the enzyme. Electrochemical detection of AA demonstrated the ability of both reduced RPLTT and C25S to chemically reduce DHA to AA in the absence of GSH. However, RPLTT had an initial rate of DHA reduction which was 4-fold greater than that of C25S, and after 10 min, RPLTT resulted in an AA concentration 11-fold greater than that of C25S. Isoelectric focusing analysis revealed that the product of reaction of reduced RPLTT but not C25S with DHA was consistent with the oxidized form of the enzyme. This result suggested that even though both RPLTT and the C25S mutant had equivalent specificity constants for DHA and GSH, they may have different catalytic mechanisms. On the basis of the experimental results, a catalytic mechanism for the DHA reductase activity of RPLTT is proposed. This is the first description of a catalytic mechanism of a glutathione:dehydroascorbate oxidoreductase (EC 1.8.5.1).

The potential of subtype-selective neuronal nicotinic acetylcholine receptor agonists as therapeutic agents.

Neuronal nicotinic acetylcholine receptors (NAChRs) are pentameric ligand-gated ion channel receptors which exist as different functional subunit combinations which apparently subserve different physiological functions as indicated by molecular biological and pharmacological techniques. It is possible to design and synthesize novel compounds that have greater selective affinities and efficacies than nicotine for different NAChRs, which should translate into different behavioral profiles and therapeutic potentials. Examples of NAChR agonists studied are nicotine, SIB-1508Y, SIB-1553A and epibatidine. These compounds have different degrees of selectivity for human recombinant NAChRs, different neurotransmitter release profiles in vitro and in vivo and differential behavioral profiles. Preclinical studies suggest that SIB-1508Y is a candidate for the treatment of the motor and cognitive deficits of Parkinson's disease, whereas SIB-1553A appears to have potential as a candidate for the treatment of Alzheimer's disease. Epibatidine has a strong analgesic profile, however the ratio between pharmacological activity and undesirable effects is so low that it is difficult to envisage the use of this compound therapeutically. Nicotine has a broad profile of pharmacological activity, for instance demonstrating activity in models for cognition and analgesia. As for epibatidine, the adverse effects of nicotine severely limits its therapeutic use in humans. The discovery of subtype-selective NAChR agonists such as SIB-1508Y and SIB-1553A provides a new class of neuropsychopharmacological agents with better therapeutic ratios than nonspecific agents such as nicotine.