Showing papers by "Oregon Health & Science University published in 1993"

Phosphorylated CREB binds specifically to the nuclear protein CBP

Abstract: Cyclic AMP-regulated gene expression frequently involves a DNA element known as the cAMP-regulated enhancer (CRE). Many transcription factors bind to this element, including the protein CREB, which is activated as a result of phosphorylation by protein kinase A. This modification stimulates interaction with one or more of the general transcription factors or, alternatively, allows recruitment of a co-activator. Here we report that CREB phosphorylated by protein kinase A binds specifically to a nuclear protein of M(r) 265K which we term CBP (for CREB-binding protein). Fusion of a heterologous DNA-binding domain to the amino terminus of CBP enables the chimaeric protein to function as a protein kinase A-regulated transcriptional activator. We propose that CBP may participate in cAMP-regulated gene expression by interacting with the activated phosphorylated form of CREB.

Destabilization of tracts of simple repetitive DNA in yeast by mutations affecting DNA mismatch repair

Abstract: The genomes of all eukaryotes contain tracts of DNA in which a single base or a small number of bases is repeated. Expansions of such tracts have been associated with several human disorders including the fragile X syndrome. In addition, simple repeats are unstable in certain forms of colorectal cancer, suggesting a defect in DNA replication or repair. We show here that mutations in any three yeast genes involved in DNA mismatch repair (PMS1, MLH1 and MSH2) lead to 100- to 700-fold increases in tract instability, whereas mutations that eliminate the proof-reading function of DNA polymerases have little effect. The meiotic stability of the tracts is similar to the mitotic stability. These results suggest that tract instability is associated with DNA polymerases slipping during replication, and that some types of colorectal cancer may reflect mutations in genes involved in DNA mismatch repair.

Recent advances in the expression of foreign genes in Pichia pastoris

Abstract: The Pichia pastoris heterologous gene expression system has been utilized to produce attractive levels of a variety of intracellular and extracellular proteins of interest. Recent advances in our understanding and application of the system have improved its utility even further. These advances include: (1) methods for the construction of P. pastoris strains with multiple copies of AOX1-promoter-driven expression cassettes; (2) mixed-feed culture strategies for high foreign protein volumetric productivity rates; (3) methods to reduce proteolysis of some products in high cell-density culture media; (4) tested procedures for purification of secreted products; and (5) detailed information on the structures of N-linked oligosaccharides on P. pastoris secreted proteins. In this review, these advances along with basic features of the P. pastoris system are described and discussed.

Determining the optical properties of turbid media by using the adding–doubling method

Abstract: A method is described for finding the optical properties (scattering, absorption, and scattering anisotropy) of a slab of turbid material by using total reflection, unscattered transmission, and total transmission measurements. This method is applicable to homogeneous turbid slabs with any optical thickness, albedo, or phase function. The slab may have a different index of refraction from its surroundings and may or may not be bounded by glass. The optical properties are obtained by iterating an adding–doubling solution of the radiative transport equation until the calculated values of the reflection and transmission match the measured ones. Exhaustive numerical tests show that the intrinsic error in the method is <3% when four quadrature points are used.

Identification of a receptor for gamma melanotropin and other proopiomelanocortin peptides in the hypothalamus and limbic system.

Abstract: Corticotropin (ACTH) and melanotropin (MSH) peptides (melanocortins) are produced not only in the pituitary but also in the brain, with highest concentrations in the arcuate nucleus of the hypothalamus and the commisural nucleus of the solitary tract. We have identified a receptor for MSH and ACTH peptides that is specifically expressed in regions of the hypothalamus and limbic system. This melanocortin receptor (MC3-R) is found in neurons of the arcuate nucleus known to express proopiomelanocortin (POMC) and in a subset of the nuclei to which these neurons send projections. The MC3-R is 43% identical to the MSH receptor present in melanocytes and is strongly coupled to adenylyl cyclase. Unlike the MSH or ACTH receptors, MC3-R is potently activated by gamma-MSH peptides, POMC products that were named for their amino acid homology with alpha- and beta-MSH, but lack melanotropic activity. The primary biological role of the gamma-MSH peptides is not yet understood. The location and properties of this receptor provide a pharmacological basis for the action of POMC peptides produced in the brain and possibly a specific physiological role for gamma-MSH.

beta-Amyloid-(1-42) is a major component of cerebrovascular amyloid deposits: implications for the pathology of Alzheimer disease

Abstract: Reinvestigation of the chemical structure of beta-amyloid peptide (A beta) deposits in the vascular tissue of Alzheimer disease brains revealed that the 42-residue form A beta-(1-42), rather than the more soluble A beta-(1-40) form, is the predominant peptide. Following removal of the surrounding tissue with SDS and collagenase, A beta was solubilized in formic acid and purified by Superose 12 chromatography. Peptides generated by enzymatic and chemical digestion of the A beta were purified by HPLC and characterized by amino acid analysis, sequence analysis, and mass spectrometry. In the leptomeningeal vessels, the average ratio of A beta-(1-42)/A beta-(1-40) was 58:42, whereas in the parenchymal vessels this ratio was 75:25. Interestingly, vascular A beta contains considerably less isomerized and racemized aspartyl residues than does neuritic plaque A beta, suggesting that the vascular amyloid is "younger." The discrete nature of the bands and spherical deposits of A beta associated with arterioles and capillaries, respectively, suggests that this amyloid arises from the vascular tissue itself. Increasing A beta deposition appears to lead to the distortion and occlusion of capillaries, which may contribute significantly to the pathology of Alzheimer disease.

Molecular Diversity of the Dopamine Receptors

Abstract: Our current understanding of the relationship between the dopaminergic system and human brain disorders is based on two fundamental discoveries: dopamine-replacement therapy can alleviate Parkinson’s disease (1-3) and, secondly, many antipsychotic drugs are dopamine receptor antagonists (4-7). These discoveries have guided two major directions in dopamine-related basic research and drug design: to activate dopamine r ceptors left understimulated by the degeneration of the afferent dopamine-secreting cells and to prevent dopamine from binding to its receptor, according to the hypothesis that schizophrenia is the result of dopamine receptor overactivity (5, 8). Since blockade of the dopamine r ceptors (antipsychotic therapy) can lead to a state similar to that resulting from dopamine depletion (Parkinson’s therapy) and higher doses of dopamine can cause psychoses, the therapies of disorders resulting from dopamine imbalances are associated with adverse side effects. The ideal drug(s) that will treat one disorder without affecting the other has thus far not been found. However, the search for such a drug has led to the design of several dopamine r ceptor ligands that, in turn, have increased our understanding of the dopaminergic system. In particular, these studies have

Nonuniform probability of glutamate release at a hippocampal synapse

Abstract: A change in the probability of neurotransmitter release (Pr) is an important mechanism underlying synaptic plasticity. Although Pr is often assumed to be the same for all terminals at a single synapse, this assumption is difficult to reconcile with the nonuniform size and structure of synaptic terminals in the central nervous system. Release probability was measured at excitatory synapses on cultured hippocampal neurons by analysis of the progressive block of N-methyl-D-aspartate receptor-mediated synaptic currents by the irreversible open channel blocker MK-801. Release probability was nonuniform (range of 0.09 to 0.54) for terminals arising from a single axon, the majority of which had a low Pr. However, terminals with high Pr are more likely to be affected by the activity-dependent modulation that occurs in long-term potentiation.

Voluntary consumption of ethanol in 15 inbred mouse strains

Abstract: To determine genetic differences in ethanol consumption, 15 commonly used inbred strains of mice were given ad libitum two-bottle choice between ethanol, 0.2% saccharin, or ethanol plus saccharin in one bottle versus tap water in the other bottle. Three different concentrations of ethanol were used: 3%, 6% and 10% (v/v). Of the 15 strains, the C57BL/6J, C57BR/cdJ and C57L/J strains showed the most consistent higher intake of ethanol either with or without 0.2% saccharin. In marked contrast, the DBA/1J and DBA/2J strains consistently showed the lowest intake. Consumption of 3% ethanol without saccharin was highly genetically correlated with saccharin consumption (r=0.77), suggesting that low concentrations of ethanol may have a sweet taste that affects voluntary consumption. Most strains showed very different patterns of response to ethanol with or without saccharin. Three patterns of strain responses were identified. Some strains avoided higher concentrations of ethanol whether in water or saccharin; some appeared to be sensitive to the ability of saccharin to mask the odor of ethanol; and some may have reduced consumption only when ethanol concentrations were high enough to produce aversive postingestional effects. Whereas earlier studies generally attempted to explain strain differences in consumption by invoking a single mechanism, our results demonstrate that more than one mechanism is necessary to explain the preferential ethanol intake of all strains studied.

A Decision Analysis of Alternative Treatment Strategies for Clinically Localized Prostate Cancer

Abstract: Objective. —To model the impact of initial therapy on outcomes for men with localized (clinical stage A or B) prostatic carcinoma. Design. —A decision analysis modeling three strategies: radical prostatectomy, external-beam radiation therapy, and watchful waiting, with delayed hormonal therapy if metastatic disease develops. We modeled the main benefit of treatment as a reduction in the chance of death or disutility from metastatic disease. These benefits were offset in the model by the risks of treatment-related morbidity and mortality. The model was used to analyze expected outcomes by tumor grade (well, moderately, and poorly differentiated) for men 60 to 75 years of age. Data. —Probabilities and rates for important clinical events, obtained through review of the literature for prostatic carcinoma and analysis of Medicare claims data. Main Results. —Several patterns emerged within the range of uncertainty about the risks and benefits of treatment for prostatic carcinoma. In patients with well-differentiated tumor grades, based on clinical staging, treatment at best offers limited benefit in terms of quality-adjusted life expectancy and may result in harm to the patient. Among patients with moderately or poorly differentiated tumors, if we use the most optimistic assumptions about treatment efficacy, then patients aged 60 to 65 years would benefit from either radical prostatectomy or external-beam radiation therapy, compared with watchful waiting. However, in most other cases, treatment offers less than a 1-year improvement in quality-adjusted life expectancy or decreases the quality-adjusted life expectancy compared with watchful waiting. Invasive treatment generally appears to be harmful for patients older than 70 years. Conclusions. —Radical prostatectomy and radiation therapy may benefit selected groups of patients with localized prostate cancer, particularly younger patients with higher-grade tumors. However, our model shows that in most cases the potential benefits of therapy are small enough that the choice of therapy is sensitive to the patient's preferences for various outcomes and discounting. The choice of watchful waiting is a reasonable alternative to invasive treatment for many men with localized prostatic carcinoma. (JAMA. 1993;269:2650-2658)

Efficacy of aerosolized tobramycin in patients with cystic fibrosis.

Abstract: Background Direct aerosol delivery of aminoglycosides such as tobramycin to the lower airways of patients with cystic fibrosis may control infection with Pseudomonas aeruginosa and improve pulmonary function, with low systemic toxicity. We conducted a randomized crossover study to evaluate the safety and efficacy of aerosolized tobramycin in patients with cystic fibrosis and P. aeruginosa infections. Methods Seventy-one patients with stable pulmonary status were recruited from seven U.S. centers for the treatment of cystic fibrosis and randomly assigned to one of two crossover regimens. Group 1 received 600 mg of aerosolized tobramycin for 28 days, followed by half-strength physiologic saline (placebo) for two 28-day periods. Group 2 received placebo for 28 days, followed by tobramycin for two 28-day periods. Pulmonary function, the density of P. aeruginosa in sputum, ototoxicity, nephrotoxicity, and the emergence of tobramycin-resistant P. aeruginosa were monitored. Results In the first 28-day period, tr...

Phosphorylation and regulation of glutamate receptors by calcium/calmodulin-dependent protein kinase II

Abstract: The major postsynaptic density (PSD) protein at glutaminergic synapses is calcium/calmodulin-dependent protein kinase II (CaM-K II), but its function in the PSD is not known. We have examined glutamate receptors (GluRs) as substrates for CaM-K II because (1) they are colocalized in the PSD, (2) cloned GluRs contain consensus phosphorylation sites for protein kinases including CaM-K II, and (3) several GluRs are regulated by other protein kinases. Regulation of GluRs, which are involved in excitatory synaptic transmission and in mechanisms of learning and memory, by CaM-K II is of interest because of the postulated role of CaM-K II in synaptic plasticity and its known involvement in induction of long-term potentiation. Furthermore, mice lacking the major neural isoform of CaM-K II exhibit deficits in models of learning and memory that require hippocampal input. We report here that CaM-K II phosphorylates GluR in several in vitro systems, including the PSD, and that activated CaM-K II enhances kainate-induced ion current three- to fourfold in cultured hippocampal neurons. These results are consistent with a role for PSD CaM-K II in strengthening postsynaptic GluR responses in synaptic plasticity.

The Phenomenology of Knowing the Patient

Abstract: Nurses' discourse about knowing the patient emerged as a recurring theme in an interpretive phenomenological study of the development of expertise in critical care nursing. The purpose of this article is to present analyses related to the meaning of knowing the patient, and its role in everyday nursing practice. Informants in the study were 130 nurses who practiced in adult, pediatric and newborn intensive care units of eight hospitals in three metropolitan areas. The data were group interviews in which nurses gave narrative accounts of exemplars from their practice; in addition, a sub-sample of 48 nurses were observed in their practice and participated in intensive personal history interviews. Knowing the patient means both knowing the patient's typical pattern of responses and knowing the patient as a person. Knowing the patient is central to skilled clinical judgment, requires involvement, and sets up the possibility for patient advocacy and for learning about patient populations.

Inactivation of NMDA channels in cultured hippocampal neurons by intracellular calcium

Abstract: Calcium-dependent inactivation of NMDA channels was examined on cultured rat hippocampal neurons using whole-cell voltage-clamp and cell-attached single-channel recording. An ATP regeneration solution was included in the patch pipette to retard current "rundown." In normal [Ca2+]o (1-2 mM) and 10 microM glycine, macroscopic currents evoked by 15 sec applications of NMDA (10 microM) inactivated slowly following an initial peak. At -50 mV in cells buffered to [Ca2+]i < 10(-8) M with 10 mM EGTA, the inactivation time constant (tau inact) was approximately 5 sec. Inactivation did not occur at membrane potentials of +40 mV and was absent at [Ca2+]o < or = 0.2 mM, suggesting that inactivation resulted from transmembrane calcium influx. The percentage inactivation and tau inact were dependent on [Ca2+]o. The tau inact was also longer with BAPTA in the whole-cell pipette compared to EGTA, suggesting that tau inact reflects primarily the rate of accumulation of intracellular calcium. Inactivation was incomplete, reaching a steady state level of 40-50% of the peak current. At steady state, block of open NMDA channels with MK-801 ((+)-5-methyl-10,11-dihydro-5H- dibenzo[a,d]cyclohepten-5,10-imine) completely blocked subsequent responses to NMDA, suggesting that "inactivated" channels can reopen at steady state. Inactivation was fully reversible in the presence of ATP but was not blocked by inhibiting phosphatases or proteases. In cell-attached patches, transient increases in [Ca2+]i following cell depolarization also resulted in inactivation of NMDA channels without altering the single-channel conductance. This suggests that Ca(2+)-dependent inactivation occurs in intact cells and can be triggered by calcium entry through nearby voltage-gated calcium channels, although calcium entry through NMDA channels was more effective. We suggest that [Ca2+]i transients induce NMDA channel inactivation by binding to either the channel or a nearby regulatory protein to alter channel gating. This mechanism may play a role in downregulation of postsynaptic calcium entry during sustained synaptic activity.

Control of nonlinear dynamical systems using neural networks: controllability and stabilization

Abstract: An attempt is made to indicate how practically viable controllers can be designed using neural networks, based on results in nonlinear control theory. The problem of stabilization of a dynamical system around an equilibrium point when the state of the system is accessible is considered. Simulation results are included to complement the theoretical discussions. >

Differences in coronary mortality can be explained by differences in cholesterol and saturated fat intakes in 40 countries but not in France and Finland. A paradox.

Abstract: BACKGROUNDFor decades, the coronary heart disease (CHD) mortality rate has been four or more times higher in Finland than in France despite comparable intakes of dietary cholesterol and saturated fat. A potential answer to this paradox is provided by this study of 40 countries and the analyses of other nutrients in the diets besides cholesterol and saturated fat.METHODS AND RESULTSCHD death rates for men aged 55 to 64 years were derived from the World Health Organization annual vital statistics. Dietary intakes were gathered from the Food and Agriculture Organization of the United Nations database. Forty countries at various levels of economic development and 40 dietary variables were investigated, including a lipid score that combined the intakes of cholesterol and saturated fat (Cholesterol-Saturated Fat Index [CSI]). The CSI was significantly and positively related to CHD mortality in the 40 countries. The countries with low CSIs had low CHD death rates. Countries with high CSIs had a wide range of CHD...

Loss of fumarylacetoacetate hydrolase is responsible for the neonatal hepatic dysfunction phenotype of lethal albino mice.

Abstract: Mice homozygous for the c14CoS albino deletion die as neonates as a result of liver dysfunction. Previous mapping studies have associated this defect with a 310-kb fragment encoding the hepatocyte-specific developmental regulation locus (alf/hsdr-1). The gene encoding fumarylacetoacetate hydrolase (Fah), a metabolic enzyme that catalyzes the last step of tyrosine catabolism, also maps to the same deletion interval. To test whether the neonatal defects found in the albino deletion mutants are attributable to loss of Fah, and not to another gene mapping to the deletion, we have generated Fah mutant mice by gene targeting in embryonic stem cells. Fah-deficient mice die within 12 hr after birth from hypoglycemia and liver dysfunction. In addition, the same pattern of altered liver mRNA expression found in the albino deletion mutants was also found in affected animals. We conclude that the neonatal lethal and liver dysfunction phenotype of the alf/hsdr-1 deletion is entirely attributable to loss of Fah.

The rapid detection of unknown mutations in nucleic acids.

Abstract: The task of identifying mutations in nucleic acid sequences is a vital component of research in mammalian genetics. With the advent of the polymerase chain reaction, several useful mutation detection techniques have evolved in recent years. The different methods have complementing strengths and a suitable procedure for virtually any experimental situation is now available.

Fibromyalgia and quality of life: a comparative analysis.

Abstract: The quality of life of women with fibromyalgia was explored and compared to the quality of life of women with rheumatoid arthritis, osteoarthritis, permanent ostomies, chronic obstructive pulmonary disease, insulin dependent diabetes, and healthy controls. The women with fibromyalgia consistently scored among the lowest in all domains measured. These results suggest that fibromyalgia may adversely affect quality of life to an extent not previously recognized.

Substance P selectively activates TNF-alpha gene expression in murine mast cells.

Abstract: There is increasing evidence that the neurologic system is capable of modulating a wide range of immunologic responses, including certain inflammatory processes in the lung, gastrointestinal tract, and skin. It has been proposed that secreted neuropeptides such as substance P (SP) may mediate these neuroinflammatory interactions by binding to and stimulating immune cells such as mast cells and lymphoid cells. SP is secreted in a variety of tissues by an extensive network of neurosensory C and A5 fibers in response to a wide range of noxious stimuli and injury. Previous studies to examine the effect of SP on mast cells have focused on its role in triggering histamine release and mediating immediate hypersensitivity responses. Recently it was demonstrated that mast cells are also capable of secreting multiple cytokines including TNF-alpha, IL-1, IL-3, IL-4, IL-6, and GM-CSF. In this study we tested the possibility that SP may also influence mast cell-mediated late inflammatory events by modulating the production of one or several of these cytokines. Our results indicate that SP induces TNF-alpha mRNA expression and TNF-alpha secretion in a dose-dependent manner in a murine mast cell line, CFTL12. Likewise, SP stimulates TNF-alpha secretion in freshly isolated murine peritoneal mast cells. The induction of mast cell TNF-alpha is selective, since SP does not stimulate the production of IL-1, IL-3, IL-4, IL-6, or GM-CSF in these cells. The CFTL 12 mast cell line constitutively expresses high levels of SP receptor mRNA which is not modulated by PMA/cycloheximide treatment or SP. These results further support the concept that the neurologic system modulates inflammatory events by neuropeptide-mediated mast cell cytokine release.

Renal renin-angiotensin system in diabetes: Functional, immunohistochemical, and molecular biological correlations

Abstract: Recent evidence indicates a role for the renin-angiotensin system (RAS) in the pathogenesis of glomerular injury in diabetes. To further explore the RAS in diabetes, studies were conducted in nondiabetic control rats and in moderately hyperglycemic diabetic (DM) rats. In DM rats, both acute and chronic therapy with the specific angiotensin II (ANG II) receptor antagonist losartan did not affect glomerular hyperfiltration or hyperperfusion but selectively normalized the glomerular capillary hydraulic pressure and ultrafiltration coefficient. To determine the basis of intrarenal hemodynamic responsiveness to RAS inhibition, we conducted biochemical, molecular biological, and immunohistochemical studies to assess endogenous RAS activity. Values for plasma renin concentration and serum angiotensin-converting enzyme (ACE) activity in DM rats were normal. In contrast, intrarenal renin protein content, and renin and angiotensinogen mRNAs, were increased in DM rats, suggesting disproportionate activation of the intrarenal RAS. Total renal ACE activity was significantly reduced in DM rats, but immunohistochemical studies indicated redistribution of ACE in the diabetic kidney. Proximal tubule ACE activity was reduced, but ACE immunostaining intensity was enhanced in glomeruli and renal vasculature. Together, these observations indicate increased RAS activity in those sites (glomeruli and vasculature) most likely to regulate hemodynamic function, potentially explaining the prominent responses to pharmacological blockade of ANG II formation and/or action.