Showing papers by "Oregon Health & Science University published in 1994"

Mutation in the DNA mismatch repair gene homologue hMLH 1 is associated with hereditary non-polyposis colon cancer

Abstract: The human DNA mismatch repair gene homologue hMSH2, on chromosome 2p is involved in hereditary non-polyposis colon cancer (HNPCC) On the basis of linkage data, a second HNPCC locus was assigned to chromosome 3p21-23 (ref 3) Here we report that a human gene encoding a protein, hMLH1 (human MutL homologue), homologous to the bacterial DNA mismatch repair protein MutL, is located on human chromosome 3p213-23 We propose that hMLH1 is the HNPCC gene located on 3p because of the similarity of the hMLH1 gene product to the yeast DNA mismatch repair protein, MLH1, the coincident location of the hMLH1 gene and the HNPCC locus on chromosome 3, and hMLH1 missense mutations in affected individuals from a chromosome 3-linked HNPCC family

RASTA processing of speech

Abstract: Performance of even the best current stochastic recognizers severely degrades in an unexpected communications environment. In some cases, the environmental effect can be modeled by a set of simple transformations and, in particular, by convolution with an environmental impulse response and the addition of some environmental noise. Often, the temporal properties of these environmental effects are quite different from the temporal properties of speech. We have been experimenting with filtering approaches that attempt to exploit these differences to produce robust representations for speech recognition and enhancement and have called this class of representations relative spectra (RASTA). In this paper, we review the theoretical and experimental foundations of the method, discuss the relationship with human auditory perception, and extend the original method to combinations of additive noise and convolutional noise. We discuss the relationship between RASTA features and the nature of the recognition models that are required and the relationship of these features to delta features and to cepstral mean subtraction. Finally, we show an application of the RASTA technique to speech enhancement. >

Nuclear protein CBP is a coactivator for the transcription factor CREB

Abstract: The transcription factor CREB binds to a DNA element known as the cAMP-regulated enhancer (CRE). CREB is activated through phosphorylation by protein kinase A (PKA), but precisely how phosphorylation stimulates CREB function is unknown. One model is that phosphorylation may allow the recruitment of coactivators which then interact with basal transcription factors. We have previously identified a nuclear protein of M(r)265K, CBP, that binds specifically to the PKA-phosphorylated form of CREB. We have used fluorescence anisotropy measurements to define the equilibrium binding parameters of the phosphoCREB:CBP interaction and report here that CBP can activate transcription through a region in its carboxy terminus. The activation domain of CBP interacts with the basal transcription factor TFIIB through a domain that is conserved in the yeast coactivator ADA-1 (ref. 8). Consistent with its role as a coactivator, CBP augments the activity of phosphorylated CREB to activate transcription of cAMP-responsive genes.

Sunburn and p53 in the onset of skin cancer

Abstract: Squamous cell carcinoma of the skin (SCC) can progress by stages: sun-damaged epidermis, with individual disordered keratinocytes; actinic keratosis (AK), spontaneously regressing keratinized patches having aberrant cell differentiation and proliferation; carcinoma in situ; SCC and metastasis. To understand how sunlight acts as a carcinogen, we determined the stage at which sunlight mutates the p53 tumour-suppressor gene and identified a function for p53 in skin. The p53 mutations induced by ultraviolet radiation and found in > 90% of human SCCs were present in AKs. Inactivating p53 in mouse skin reduced the appearance of sunburn cells, apoptotic keratinocytes generated by overexposure to ultraviolet. Skin thus appears to possess a p53-dependent 'guardian-of-the-tissue' response to DNA damage which aborts precancerous cells. If this response is reduced in a single cell by a prior p53 mutation, sunburn can select for clonal expansion of the p53-mutated cell into the AK. Sunlight can act twice: as tumour initiator and tumour promoter.

Reductive Dehalogenation of Chlorinated Methanes by Iron Metal

Abstract: Reduction of chlorinated solvents by fine-grained iron metal was studied in well-mixed anaerobic batch systems in order to help assess the utility of this reaction in remediation of contaminated groundwater. Iron sequentially dehalogenates carbon tetrachloride via chloroform to methylene chloride. The initial rate of each reaction step was pseudo-first-order in substrate and became substantially slower with each dehalogenation step. Thus, carbon tetrachloride degradation typically occurred in several hours, but no significant reduction of methylene chloride was observed over 1 month. Trichloroethene (TCE) was also dechlorinated by iron, although more slowly than carbon tetrachloride. Increasing the clean surface area of iron greatly increased the rate of carbon tetrachloride dehalogenation, whereas increasing pH decreased the reduction rate slightly. The reduction of chlorinated methanes in batch model systems appears to be coupled with oxidative dissolution (corrosion) of the iron through a largely diffusion-limited surface reaction.

Localization of the melanocortin-4 receptor (MC4-R) in neuroendocrine and autonomic control circuits in the brain.

Abstract: POMC, the precursor of ACTH, MSH, and beta-endorphin peptides, is expressed in the pituitary and in two sites in the brain, in the arcuate nucleus of the hypothalamus and the commissural nucleus of the solitary tract of the brain stem. Little is known regarding the functions of melanocortin (ACTH and MSH) peptides in the brain. We report here the detailed neuroanatomical distribution of the MC4-R mRNA in the adult rat brain. The melanocortin 3 receptor (MC3-R), characterized previously, was found to be expressed in arcuate nucleus neurons and in a subset of their presumptive terminal fields but in few regions of the brainstem. The highly conserved MC4-R is much more widely expressed than MC3-R and is pharmacologically distinct. MC4-R mRNA was found in multiple sites in virtually every brain region, including the cortex, thalamus, hypothalamus, brainstem, and spinal cord. Unlike the MC3-R, MC4-R mRNA is found in both parvicellular and magnocellular neurons of the paraventricular nucleus of the hypothalamus...

Agouti protein is an antagonist of the melanocyte-stimulating-hormone receptor.

Abstract: The genetic loci agouti and extension control the relative amounts of eumelanin (brown-black) and phaeomelanin (yellow-red) pigments in mammals: extension encodes the receptor for melanocyte-stimulating hormone (MSH) and agouti encodes a novel 131-amino-acid protein containing a signal sequence. Agouti, which is produced in the hair follicle, acts on follicular melanocytes to inhibit alpha-MSH-induced eumelanin production, resulting in the subterminal band of phaeomelanin often visible in mammalian fur. Here we use partially purified agouti protein to demonstrate that agouti is a high-affinity antagonist of the MSH receptor and blocks alpha-MSH stimulation of adenylyl cyclase, the effector through which alpha-MSH induces eumelanin synthesis. Agouti was also found to be an antagonist of the melanocortin-4 receptor, a related MSH-binding receptor. Consequently, the obesity caused by ectopic expression of agouti in the lethal yellow (Ay) mouse may be due to the inhibition of melanocortin receptor(s) outside the hair follicle.

Functional comparisons of three glutamate transporter subtypes cloned from human motor cortex

Abstract: Reuptake plays an important role in regulating synaptic and extracellular concentrations of glutamate. Three glutamate transporters expressed in human motor cortex, termed EAAT1, EAAT2, and EAAT3 (for excitatory amino acid transporter), have been characterized by their molecular cloning and functional expression. Each EAAT subtype mRNA was found in all human brain regions analyzed. The most prominent regional variation in message content was in cerebellum where EAAT1 expression predominated. EAAT1 and EAAT3 mRNAs were also expressed in various non- nervous tissues, whereas expression of EAAT2 was largely restricted to brain. The kinetic parameters and pharmacological characteristics of transport mediated by each EAAT subtype were determined in transfected mammalian cells by radio-label uptake and in microinjected oocytes by voltage-clamp measurements. The affinities of the EAAT subtypes for L- glutamate were similar, with Km determinations varying from 48 to 97 microM in the mammalian cell assay and from 18 to 28 microM in oocytes. Glutamate uptake inhibitors were used to compare the pharmacologies of the EAAT subtypes. The EAAT2 subtype was distinguishable from the EAAT1/EAAT3 subtypes by the potency of several inhibitors, but most notably by sensitivity to kainic acid (KA) and dihydrokainic acid (DHK). KA and DHK potently inhibited EAAT2 transport, but did not significantly affect transport by EAAT1/EAAT3. Using voltage-clamp measurements, most inhibitors were found to be substrates that elicited transport currents. In contrast, KA and DHK did not evoke currents and they were found to block EAAT2-mediated transport competitively. This selective interaction with the EAAT2 subtype could be a significant factor in KA neurotoxicity. These studies provide a foundation for understanding the role of glutamate transporters in human excitatory neurotransmission and in neuropathology.

OHSUMED: an interactive retrieval evaluation and new large test collection for research

Abstract: A series of information retrieval experiments was carried out with a computer installed in a medical practice setting for relatively inexperienced physician end-users. Using a commercial MEDLINE product based on the vector space model, these physicians searched just as effectively as more experienced searchers using Boolean searching. The results of this experiment were subsequently used to create a new large medical test collection, which was used in experiments with the SMART retrieval system to obtain baseline performance data as well as compare SMART with the other searchers.

Major Depression in Community Adolescents: Age at Onset, Episode Duration, and Time to Recurrence

Abstract: Objective This paper presents retrospective and prospective data regarding time course parameters of major depressive disorder (MDD) in community adolescents (14 to 18 years old): time to onset and recovery and, among those who recovered, time to recurrence. Method Diagnostic interviews were conducted with 1,508 randomly selected high school students. Three hundred sixty-two had experienced at least one past or current episode of MDD. Results Mean age at onset of first episode was 14.9 (SD = 2.8). Early MDD onset was associated with female gender and suicidal ideation. MDD episode duration ranged from 2 to 520 weeks, with a mean of 26.4 weeks (SE = 3.3) and a median of 8.0 weeks. Longer episodes were observed in those whose depression occurred early (at or before age 15), whose depression had been accompanied by suicidal ideation, and for whom treatment was sought. Of the adolescents who recovered, 5% relapsed within 6 months, 12% within 1 year, and approximately 33% within 4 years. Shorter time to recurrence was associated with prior suicidal ideation and attempt and with later first onset. Conclusions Risk of MDD is low in childhood, increasing substantially with adolescence. The majority of episodes in community adolescents are relatively brief, although the risk of recurrence is substantial. Suicidal behaviors are important mediators of episode duration and of recurrence.

Differential activation of CREB by Ca2+/calmodulin-dependent protein kinases type II and type IV involves phosphorylation of a site that negatively regulates activity.

Abstract: The cAMP response element-binding protein (CREB) has been shown to mediate transcriptional activation of genes in response to both cAMP and calcium influx signal transduction pathways. The roles of two multifunctional calcium/calmodulin-dependent protein kinases, CaMKIV and CaMKII, were examined in transient transfection studies that utilized either the full-length or the constitutively active forms of these kinases. The results indicate that CaMKIV is much more potent than CaMKII in activating CREB in three different cell lines. It was also found in these studies that Ser*^^ of CREB is essential for its activation by CaMKIV. Because both CaMKII and CaMKIV can phosphorylate CREB, we pursued further the mechanism by which CaMKII and CaMKIV differentially regulate CREB activity. Mutagenesis studies and phosphopeptide mapping analysis demonstrated that in vitro, CaMKIV phosphorylates CREB at Ser^^^ only, whereas CaMKII phosphorylates CREB at Ser^^^ and a second site, Ser^*^. Transient transfection studies revealed that phosphorylation of Ser^*^ by CaMKII blocks the activation of CREB that would otherwise occur when Ser^^^ is phosphorylated. When Ser^*^ was mutated to alanine, CREB was activated by CaMKII, as well as by CaMKIV. Furthermore, mutation of Ser^*^ to alanine enhanced the ability of Ca^"^ influx to activate CREB, suggesting a physiological role for the phosphorylation of Ser^*^ in modulation of CREB activity. These data provide evidence for a new mechanism for regulation of CREB activity involving phosphorylation of a negative regulatory site in the transcriptional activation domain. The studies also provide new insights into possible interactions between the cAMP and Ca^"^ signaling pathways in the regulation of transcription. In particular, changes in intracellular Ca^"^ have the potential to either inhibit or augment the ability of cAMP to stimulate transcription, depending on the presence of specific forms of Ca^'^/calmodu lin-dependent protein kinases.

Episodic ataxia/myokymia syndrome is associated with point mutations in the human potassium channel gene, KCNA1.

Abstract: Episodic ataxia (EA) is a rare, familial disorder producing attacks of generalized ataxia, with normal or near-normal neurological function between attacks. One type of EA is characterized by brief episodes of ataxia with myokymia (rippling of muscles) evident between attacks. Linkage studies in four such families suggested localization of an EA/myokymia gene near the voltage gated K+ channel gene, KCNA1 (Kv1.1), on chromosome 12p. Mutation analysis of the KCNA1 coding region in these families identified four different missense point mutations present in the heterozygous state, indicating that EA/myokymia can result from mutations in this gene.

Neurobehavioral sequelae of cranial irradiation in adults: a review of radiation-induced encephalopathy.

Abstract: PURPOSETo examine behavioral dimensions of treatment outcomes for patients receiving cranial irradiation. Radiation encephalopathy is one of these and refers to significant cognitive and emotional dysfunction following radiation therapy to the brain. Issues of definition, estimated incidence, pathophysiologic mechanisms, and recommended research designs are reviewed in relationship to functional neurobehavioral outcomes.PATIENTS AND METHODSTwenty-nine studies of adults receiving therapeutic cranial irradiation (TCI) involving 748 patients and 18 studies of prophylactic cranial irradiation (PCI) involving 368 patients are reviewed. Assessment of patient outcomes are summarized for research published since 1980, with specific attention to adverse changes in cognitive and emotional functioning.RESULTSAnalyses revealed that 213 TCI patients and 100 PCI patients showed encephalopathy attributed to radiation. Manifestations of the late delayed effects of radiotherapy on brain function are related to patient age...

Elution of leachable components from composites.

Abstract: A significant amount of residual monomer or short chain polymers remain unbound in set composite material. Due to its potential impact on both the biocompatibility and the structural stability of the restoration, many investigators have studied the elution of these unbound molecules into aqueous media. The results of these studies suggest that elution of leachable components from composites is rapid, with the majority being released within a matter of hours. Weight losses of up to 2% of the mass of the composite have been reported under certain conditions. The studies have also shown that the extent and rate of elution of components from composites is dependent upon several factors. The quantity of leachables has been correlated to the degree of cure of the polymer network. The composition and solubility characteristics of the extraction solvent influence the kinetics and mechanism of the elution process. Elution is generally thought to occur via diffusion of molecules through the resin matrix, and is therefore dependent upon the size and chemical characteristics of the leachable species.

Preventing Falls Among Community-Dwelling Older Persons: Results From a Randomized Trial

Abstract: A randomized trial of falls prevention program that addressed home safety, exercise, and behavioral risks was conducted with 3,182 independently living HMO members age 65 and older. The intervention decreased the odds of falling by 0.85, but only reduced the average number of falls among those who fell by 7%. The effect was strongest among men age 75 and older. The likelihood of avoiding falls requiring medical treatment was not significantly affected by the intervention. We conclude that the intervention dose was not of sufficient intensity or duration to have a marked protective effect on older persons. Future research should focus on more intensive intervention approaches because serious falls do not appear to be amendable to low-intensity environment/behavioral efforts. Language: en

Testosterone influences spatial cognition in older men.

Abstract: Testosterone plays a role in the organization of behavior during development. The authors examined whether testosterone could play a maintenance role in behavior as well. In a double-blind manner, verbal and visual memory, spatial cognition, motor speed, cognitive flexibility, and mood in a group of healthy older men who were supplemented for 3 months with testosterone were assessed. The increase in testosterone levels to 150% of baseline levels resulted in a significant enhancement of spatial cognition, but no change in any other cognitive domain was found. Testosterone supplementation influenced the endogenous production of estradiol, and estradiol was found to have an inverse relationship to spatial cognitive performance. These results suggest that testosterone supplementation can modify spatial cognition in older men; however, it is likely that this occurs through testosterone's influence on estrogen.

Cell-surface receptors for gibbon ape leukemia virus and amphotropic murine retrovirus are inducible sodium-dependent phosphate symporters.

Abstract: Cell surface receptors for gibbon ape leukemia virus (Glvr-1) and murine amphotropic retrovirus (Ram-1) are distinct but related proteins having multiple membrane-spanning regions. Distant homology with a putative phosphate permease of Neurospora crassa suggested that these receptors might serve transport functions. By expression in Xenopus laevis oocytes and in mammalian cells, we have identified Glvr-1 and Ram-1 as sodium-dependent phosphate symporters. Two-electrode voltage-clamp analysis indicates net cation influx, suggesting that phosphate is transported with excess sodium ions. Phosphate uptake was reduced by > 50% in mouse fibroblasts expressing amphotropic envelope glycoprotein, which binds to Ram-1, indicating that Ram-1 is a major phosphate transporter in these cells. RNA analysis shows wide but distinct tissue distributions, with Glvr-1 expression being highest in bone marrow and Ram-1 in heart. Overexpression of Ram-1 severely repressed Glvr-1 synthesis in fibroblasts, suggesting that transporter expression may be controlled by net phosphate accumulation. Accordingly, depletion of extracellular phosphate increased Ram-1 and Glvr-1 expression 3- to 5-fold. These results suggest simple methods to modulate retroviral receptor expression, with possible applications to human gene therapy.

Length of uninterrupted CGG repeats determines instability in the FMR1 gene.

Abstract: Analysis of 84 human X chromosomes for the presence of interrupting AGG trinucleotides within the CGG repeat tract of the FMR1 gene revealed that most alleles possess two interspersed AGGs and that the longest tract of uninterrupted CGG repeats is usually found at the 3' end. Variation in the length of the repeat appears polar. Alleles containing between 34 and 55 repeats, with documented unstable transmissions, were shown to have lost one or both AGG interruptions. These comparisons define an instability threshold of 34-38 uninterrupted CGG repeats. Analysis of premutation alleles in Fragile X syndrome carriers reveals that 70% of these alleles contain a single AGG interruption. These data suggest that the loss of an AGG is an important mutational event in the generation of unstable alleles predisposed to the Fragile X syndrome.

Growth Hormone (GH) Insensitivity Due to Primary GH Receptor Deficiency

Abstract: As demonstrated in Table 2, the differential diagnosis of growth hormone insensitivity (GHI) includes a number of discrete disorders that can be broadly classified as primary or secondary forms. We have selected GHRD (Laron syndrome) as the prototypic disorder of GHI, in part because such dramatic and rapid progress has been made in this clinical condition over the last 6 yr. These advances represent the fortunate convergence of: 1) the cloning of the GHR gene and the identification of deletions and mutations of this gene in GHRD; 2) the development of assay methods for measurement of the GHBP, IGF peptides, and binding proteins; 3) the discovery of a larger number of affected individuals than had been previously suspected, including the recognition and description of a large genetically homogeneous population of GHRD patients in Ecuador; and 4) the production of recombinant IGF-I for therapeutic trials in GHRD. Although we are still in the early phases of clinical trials of recombinant hIGF-I in GHRD, preliminary results have been encouraging. Whether this promise translates into genuine improvements in height and body composition, without significant clinical toxicity, remains to be determined. Similarly, the suitability of IGF-I therapy for other, particularly secondary, forms of GHI is still uncertain, although the responsiveness of GHD-IA patients seems to parallel that seen in GHRD (66, 78). The next few years should provide exciting and potentially important new data on the clinical spectrum, biochemical and molecular characteristics, and responsiveness to therapy of syndromes of GHI.

Rapid HPLC determination of total homocysteine and other thiols in serum and plasma: sex differences and correlation with cobalamin and folate concentrations in healthy subjects.

Abstract: High-performance liquid chromatography with fluorescence detection has been utilized for the rapid determination of total homocysteine, cysteine, and cysteinylglycine in human serum and plasma. Our earlier procedure (Anal Biochem 1989;178:208), which used monobromobimane to specifically derivatize thiols, has been extensively modified to allow for rapid processing of samples. As a result, > 80 samples a day can be assayed for total homocysteine, cysteine, and cysteinylglycine. The method is sensitive (lower limit of detection < or = 4 pmol in the assay) and precise (intra- and interassay CV for homocysteine, 3.31% and 4.85%, respectively). Mean total homocysteine concentrations in plasma and serum were significantly different, both from healthy male donors (9.26 and 12.30 mumol/L, respectively; P < 0.001) and healthy female donors (7.85 and 10.34 mumol/L, respectively; P < 0.001). The differences in total homocysteine between sexes were also significant (P = 0.002 for both plasma and serum). Similar differences were found for cysteine and cysteinylglycine. We found a significant inverse correlation between serum cobalamin and total homocysteine in men (P = 0.0102) and women (P = 0.0174). Serum folate also inversely correlated with total homocysteine in both sexes.

Nucleus Tractus Solitarius—Gateway to Neural Circulatory Control

Abstract: The neural regulation of the cardiovascular system has been the subject of active investigation for more than a century. Generally, the detailed infor­ mation available about peripheral mechanisms, both afferent and efferent, far exceeds what we know about the central nervous system (CNS) pro­ cessing, which gives rise to reflex responses (80). The current picture of the CNS contribution to cardiovascular regulation consists primarily of a loose network schematic including identified cell locations and connections involved in baroreflexes (Figure 1) and is the recent topic of several excellent, comprehensive reviews (80, 88, 132, 144). Much less is known about the functional properties by which these neurons communicate, how they are modulated, or the mechanisms responsible for various features of the reflex. Over the past ten years considerable research effort has focused on the initial neurons of the baroreceptor reflex, and it is here that work is most rapidly approaching issues of the cellular basis of function. Most cardio93

Anchoring of protein kinase A is required for modulation of AMPA/kainate receptors on hippocampal neurons.

Abstract: PHOSPHORYLATION of molecules involved in synaptic transmission by multifunctional protein kinases modulates both pre- and post-synaptic events in the central nervous system1,2. The positioning of kinases near their substrates may be an important part of the regulatory mechanism. The A-kinase-anchoring proteins (AKAPs; ref. 3) are known to bind the regulatory subunit of cyclic AMP-dependent protein kinase A with nanomolar affinity4–6. Here we show that anchoring of protein kinase A by AKAPs is required for the modulation of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA)/kainate channels4,5. Intracellular per-fusion of cultured hippocampal neurons with peptides derived from the conserved kinase binding region of AKAPs prevented the protein kinase A-mediated regulation of AMPA/kainate currents as well as fast excitatory synaptic currents. This effect could be overcome by adding the purified catalytic subunit of protein kinase. A control peptide lacking kinase-binding activity had no effect. To our knowledge, these results provide the first evidence that anchoring of protein kinase A is crucial in the regulation of synaptic function.

Crystal structure of LacI member, PurR, bound to DNA: minor groove binding by alpha helices

Abstract: The three-dimensional structure of a ternary complex of the purine repressor, PurR, bound to both its corepressor, hypoxanthine, and the 16-base pair purF operator site has been solved at 2.7 A resolution by x-ray crystallography. The bipartite structure of PurR consists of an amino-terminal DNA-binding domain and a larger carboxyl-terminal corepressor binding and dimerization domain that is similar to that of the bacterial periplasmic binding proteins. The DNA-binding domain contains a helix-turn-helix motif that makes base-specific contacts in the major groove of the DNA. Base contacts are also made by residues of symmetry-related alpha helices, the "hinge" helices, which bind deeply in the minor groove. Critical to hinge helix-minor groove binding is the intercalation of the side chains of Leu54 and its symmetry-related mate, Leu54', into the central CpG-base pair step. These residues thereby act as "leucine levers" to pry open the minor groove and kink the purF operator by 45 degrees.