Showing papers by "Oregon Health & Science University published in 1996"

Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells.

Abstract: The bcr-abl oncogene, present in 95% of patients with chronic myelogenous leukemia (CML), has been implicated as the cause of this disease. A compound, designed to inhibit the Abl protein tyrosine kinase, was evaluated for its effects on cells containing the Bcr-Abl fusion protein. Cellular proliferation and tumor formation by Bcr-Abl-expressing cells were specifically inhibited by this compound. In colony-forming assays of peripheral blood or bone marrow from patients with CML, there was a 92-98% decrease in the number of bcr-abl colonies formed but no inhibition of normal colony formation. This compound may be useful in the treatment of bcr-abl-positive leukemias.

Cyclophosphamide and Cisplatin Compared with Paclitaxel and Cisplatin in Patients with Stage III and Stage IV Ovarian Cancer

Abstract: Background Chemotherapy combinations that include an alkylating agent and a platinum coordination complex have high response rates in women with advanced ovarian cancer. Such combinations provide long-term control of disease in few patients, however. We compared two combinations, cisplatin and cyclophosphamide and cisplatin and paclitaxel, in women with ovarian cancer. Methods We randomly assigned 410 women with advanced ovarian cancer and residual masses larger than 1 cm after initial surgery to receive cisplatin (75 mg per square meter of body-surface area) with either cyclophosphamide (750 mg per square meter) or paclitaxel (135 mg per square meter over a period of 24 hours). Results Three hundred eighty-six women met all the eligibility criteria. Known prognostic factors were similar in the two treatment groups. Alopecia, neutropenia, fever, and allergic reactions were reported more frequently in the cisplatin–paclitaxel group. Among 216 women with measurable disease, 73 percent in the cisplatin–pacli...

Intramuscular interferon beta-1a for disease progression in relapsing multiple sclerosis

Abstract: The accepted standard treatment of relapsing multiple sclerosis consists of medications for disease symptoms, including treatment for acute exacerbations. However, currently there is no therapy that alters the progression of physical disability associated with this disease. The purpose of this study was to determine whether interferon beta-1a could slow the progressive, irreversible, neurological disability of relapsing multiple sclerosis. Three hundred one patients with relapsing multiple sclerosis were randomized into a double-blinded, placebo-controlled, multicenter phase I11 trial of interferon beta-la. Interferon beta-la, 6.0 million units (30 μg), was administered by intramuscular injection weekly. The primary outcome variable was time to sustained disability progression of at least 1.0 point on the Kurtzke Expanded Disability Status Scale (EDSS). Interferon beta-la treatment produced a significant delay in time to sustained EDSS progression (p equals; 0.02). The Kaplan-Meier estimate of the proportion of patients progressing by the end of 104 weeks was 34.9% in the placebo group and 21.9% in the interferon beta-la-treated group. Patients treated with interferon beta-la also had significantly fewer exacerbations (p = 0.03) and a significantly lower number and volume of gadolinium-enhanced brain lesions on magnetic resonance images (pvalues ranging between 0.02 and 0.05). Over 2 years, the annual exacerbation rate was 0.90 in placebo-treated patients versus 0.61 in interferon beta-la-treated patients. There were no major adverse events related to treatment. Interferon beta- la had a significant beneficial impact in relapsing multiple sclerosis patients by reducing the accumulation of permanent physical disability, exacerbation frequency, and disease activity measured by gadolinium-enhanced lesions on brain magnetic resonance images. This treatment may alter the hndamen- tal course of relapsing multiple sclerosis.

Small-conductance, calcium-activated potassium channels from mammalian brain.

Abstract: Members of a previously unidentified family of potassium channel subunits were cloned from rat and human brain. The messenger RNAs encoding these subunits were widely expressed in brain with distinct yet overlapping patterns, as well as in several peripheral tissues. Expression of the messenger RNAs in Xenopus oocytes resulted in calcium-activated, voltage-independent potassium channels. The channels that formed from the various subunits displayed differential sensitivity to apamin and tubocurare. The distribution, function, and pharmacology of these channels are consistent with the SK class of small-conductance, calcium-activated potassium channels, which contribute to the afterhyperpolarization in central neurons and other cell types.

Flux coupling in a neuronal glutamate transporter

Abstract: Synaptic transmission is commonly terminated by diffusion and reuptake of neurotransmitter from the synaptic cleft. Glutamate reuptake prevents neurotoxicity and sets the lower limit for the concentration of extracellular glutamate, so it is important to understand the thermodynamics of this process. Here we use voltage clamping with a pH-sensitive fluorescent dye to monitor electrical currents and pH changes associated with flux of glutamate mediated by the human neuronal glutamate transporter EAAT3. In contrast to a previous model, we find that three sodium ions and one proton are cotransported with each glutamate ion into the cell, while one potassium ion is transported out of the cell. This coupling can support a transmembrane glutamate concentration gradient ([Glu]in/[Glu]out) exceeding 10(6) under equilibrium conditions, and would allow the transporter to continue removing glutamate over a wide range of ionic conditions.

Involvement of mouse Mlh1 in DNA mismatch repair and meiotic crossing over.

Abstract: Mice that are deficient in either the Pms2 or Msh2 DNA mismatch repair genes have microsatellite instability and a predisposition to tumours. Interestingly, Pms2–deficient males display sterility associated with abnormal chromosome pairing in meiosis. Here mice deficient in another mismatch repair gene, Mlh1, possess not only microsatellite instability but are also infertile (both males and females). Mlh 1 –deficient spermatocytes exhibit high levels of prematurely separated chromosomes and arrest in first division meiosis. We also show that Mlh1 appears to localize to sites of crossing over on meiotic chromosomes/Together these findings suggest that Mlh1 is involved in DNA mismatch repair and meiotic crossing over.

Crystal structure of the lactose operon repressor and its complexes with DNA and inducer.

Abstract: The lac operon of Escherichia coli is the paradigm for gene regulation. Its key component is the lac repressor, a product of the lacI gene. The three-dimensional structures of the intact lac repressor, the lac repressor bound to the gratuitous inducer isopropyl-β-D-1-thiogalactoside (IPTG) and the lac repressor complexed with a 21-base pair symmetric operator DNA have been determined. These three structures show the conformation of the molecule in both the induced and repressed states and provide a framework for understanding a wealth of biochemical and genetic information. The DNA sequence of the lac operon has three lac repressor recognition sites in a stretch of 500 base pairs. The crystallographic structure of the complex with DNA suggests that the tetrameric repressor functions synergistically with catabolite gene activator protein (CAP) and participates in the quaternary formation of repression loops in which one tetrameric repressor interacts simultaneously with two sites on the genomic DNA.

Reduction of Nitro Aromatic Compounds by Zero-Valent Iron Metal

Abstract: The properties of iron metal that make it useful in remediation of chlorinated solvents may also lead to reduction of other groundwater contaminants such as nitro aromatic compounds (NACs). Nitrobenzene is reduced by iron under anaerobic conditions to aniline with nitrosobenzene as an intermediate product. Coupling products such as azobenzene and azoxybenzene were not detected. First-order reduction rates are similar for nitrobenzene and nitrosobenzene, but aniline appearance occurs more slowly (typical pseudo-first-order rate constants 3.5 × 10-2, 3.4 × 10-2, and 8.8 × 10-3 min-1, respectively, in the presence of 33 g/L acid-washed, 18−20 mesh Fluka iron turnings). The nitro reduction rate increased linearly with concentration of iron surface area, giving a specific reaction rate constant (3.9 ± 0.2 × 10-2 min-1 m-2 L). The minimal effects of solution pH or ring substitution on nitro reduction rates, and the linear correlation between nitrobenzene reduction rate constants and the square-root of mixing ra...

X-linked anhidrotic (hypohidrotic) ectodermal dysplasia is caused by mutation in a novel transmembrane protein.

Abstract: Ectodermal dysplasias comprise over 150 syndromes of unknown pathogenesis. X-linked anhidrotic ectodermal dysplasia (EDA) is characterized by abnormal hair, teeth and sweat glands. We now describe the positional cloning of the gene mutated in EDA. Two exons, separated by a 200-kilobase intron, encode a predicted 135-residue transmembrane protein. The gene is disrupted in six patients with X;autosome translocations or submicroscopic deletions; nine patients had point mutations. The gene is expressed in keratinocytes, hair follicles, and sweat glands, and in other adult and fetal tissues. The predicted EDA protein may belong to a novel class with a role in epithelial-mesenchymal signalling.

Kinetics of Halogenated Organic Compound Degradation by Iron Metal

Abstract: A combination of new and previously reported data on the kinetics of dehalogenation by zero-valent iron (Fe0) has been subjected to an analysis of factors effecting contaminant degradation rates. First-order rate constants (kobs) from both batch and column studies vary widely and without meaningful correlation. However, normalization of these data to iron surface area concentration yields a specific rate constant (kSA) that varies by only 1 order of magnitude for individual halocarbons. Correlation analysis using kSA reveals that dechlorination is generally more rapid at saturated carbon centers than unsaturated carbons and that high degrees of halogenation favor rapid reduction. However, new data and additional analysis will be necessary to obtain reliable quantitative structure−activity relationships. Further generalization of our kinetic model has been obtained by accounting for the concentration and saturation of reactive surface sites, but kSA is still the most appropriate starting point for design c...

Coordination of Three Signaling Enzymes by AKAP79, a Mammalian Scaffold Protein

Abstract: Multivalent binding proteins, such as the yeast scaffold protein Sterile-5, coordinate the location of kinases by serving as platforms for the assembly of signaling units. Similarly, in mammalian cells the cyclic adenosine 3',5'-monophosphate-dependent protein kinase (PKA) and phosphatase 2B [calcineurin (CaN)] are complexed by an A kinase anchoring protein, AKAP79. Deletion analysis and binding studies demonstrate that a third enzyme, protein kinase C (PKC), binds AKAP79 at a site distinct from those bound by PKA or CaN. The subcellular distributions of PKC and AKAP79 were similar in neurons. Thus, AKAP79 appears to function as a scaffold protein for three multifunctional enzymes.

Tolerance of single, but not multiple, amino acid replacements in antibody VH CDR 2: a means of minimizing B cell wastage from somatic hypermutation?

Abstract: Mutations in the heavy chain complementarity determining region 2 (CDR2) of the phosphocholine-specific T15 Ab can have a dramatic effect on the ability of the Ab to bind Ag. A panel of multisite mutants that had lost detectable binding to phosphocholine-containing Ags was previously created by saturation mutagenesis of the CDR2 region of T15. Based on the predicted importance of amino acid changes represented in the multisite mutants, we have created single-site mutations, yielding a panel of Abs with which to test 17 of the 19 CDR2 residues. Of the 17 positions examined, only one, Arg52, is intolerant to change, yielding a nonbinder phenotype even with conservative amino acid replacement. Mutation at two other sites, Ala50 and Tyr55, can yield a nonbinder phenotype depending on the amino acid replacement. Single-site mutations of the remaining 14 positions allowed retention of binding ability. Thus, except for positions 50, 52, and 55, multiple mutations must be introduced into the CDR2 region to create a nonbinder phenotype. We provide a newly refined model of T15, illustrating the structure and the interactions of the CDR2 region. Our results imply that introduction of point mutations would not normally delete Ag-binding ability until two or more mutations had accumulated. This would minimize potentially harmful effects of somatic mutation on Ig V region genes and improve the chance of survival for an Ab such as T15, which in its unmutated form is already well suited to bind Ag.

Hepatocytes corrected by gene therapy are selected in vivo in a murine model of hereditary tyrosinaemia type I

Abstract: Current strategies for hepatic gene therapy are either quantitatively inefficient or suffer from lack of permanent gene expression. We have utilized an animal model of hereditary tyrosinaemia type I (HT1), a recessive liver disease caused by deficiency of fumarylacetoacetate hydrolase (FAH), to determine whether in vivo selection of corrected hepatocytes could improve the efficiency of liver gene transfer. As few as 1,000 transplanted wild-type hepatocytes were able to repopulate mutant liver, demonstrating their strong competitive growth advantage. Mutant hepatocytes corrected in situ by retroviral gene transfer were also positively selected. In mutant animals treated by multiple retrovirus injections >90% of hepatocytes became FAH positive and liver function was restored to normal. Our results demonstrate that in vivo selection is a useful strategy for hepatic gene therapy and may lead to effective treatment of human HT1 by retroviral gene transfer.

Associative learning disrupted by impaired Gs signaling in Drosophila mushroom bodies

Abstract: Disruptions in mushroom body (MB) or central complex (CC) brain structures impair Drosophila associative olfactory learning. Perturbations in adenosine 3',5' monophosphate signaling also disrupt learning. To integrate these observations, expression of a constitutively activated stimulatory heterotrimeric guanosine triphosphate-binding protein alpha subunit (Galphas*) was targeted to these brain structures. The ability to associate odors with electroshock was abolished when Galphas* was targeted to MB, but not CC, structures, whereas sensorimotor responses to these stimuli remained normal. Expression of Galphas* did not affect gross MB morphology, and wild-type Galphas expression did not affect learning. Thus, olfactory learning depends on regulated Gs signaling in Drosophila MBs.

The melanocortin receptors: agonists, antagonists, and the hormonal control of pigmentation

Abstract: Molecular cloning experiments have led to the identification and characterization of a family of five receptors for the melanocortin (melanotropic and adrenocorticotropic) peptides. The first two members of the family cloned were the well-characterized melanocyte-stimulating hormone receptor (MSH-R) and adrenocorticotropin receptor (ACTH-R). The three new melanocortin receptors have been termed the MC3-R, MC4-R, and MC5-R, according to the order of their discovery, and little is known at this point concerning their function. Agouti and extension are two genetic loci known to control the amounts of eumelanin (brown-black) and phaeomelanin (yellow-red) pigments. Chromosomal mapping demonstrated that the MSH-R, now termed MCI-R, mapped to extension. Extension was shown to encode the MCI-R, and mutations in the MCI-R are responsible for the different pigmentation phenotypes caused by this locus. Functional variants of the MCI-R, originally characterized in the mouse, have now also been identified in the guinea pig and cow. Dominant constitutive mutants of the MCI-R are responsible for causing dark black coat colors while recessive alleles result in yellow or red coat colors. Agouti, a secreted 108 amino acid peptide produced within the hair follicle, acts on follicular melanocytes to inhibit alpha-MSH-induced eumelanin production. Experiments demonstrate that agouti is a high-affinity antagonist, acting at the MCI-R to block alpha-MSH stimulation of adenylyl cyclase, the effector through which alpha-MSH induces eumelanin synthesis. The MCI-R is thus a unique bifunctionally controlled receptor, activated by alpha-MSH and antagonized by agouti, both contributing to the variability seen in mammalian coat colors. The variable tan and black coat color patterns seen in the German Shepherd, for example, can now be understood on the molecular level as the interaction of a number of extension and agouti alleles encoding variably functioning receptors and a differentially expressed antagonist of the receptor, respectively.

Altered Expression of hMSH2 and hMLH1 in Tumors with Microsatellite Instability and Genetic Alterations in Mismatch Repair Genes

Abstract: To date, at least four genes involved in DNA mismatch repair (MMR) have been demonstrated to be altered in the germline of patients with hereditary nonpolyposis colon cancer: hMSH2, hMLH1, hPMS1, and hPMS2. Additionally, loss of MMR function has been demonstrated to lead to the phenomenon of microsatellite instability (MIN) in tumors from these patients. In this study, we have examined the protein expression pattern of hMSH2 and hMLH1 by immunohistochemistry in paraffin-embedded tumors from 7 patients with MIN+ sporadic cancer, 13 patients with familial colorectal cancer, and 12 patients meeting the strict Amsterdam criteria for hereditary nonpolyposis colon cancer. The relationship between the expression of these two gene products, the presence of germline or somatic mutations, and the presence of tumor MIN was examined. Nineteen of the 28 tumors studied demonstrated MIN, whereas mutations in hMLH1 and hMSH2 were detected in 6 and 2 patients, respectively. Of the eight MIN+/mutation+ cases, the absence of protein expression was observed for the corresponding gene product in all but one case (missense mutation in hMLH1). However, seven MIN+/mutation- cases also showed no expression of either hMLH1 (n = 5), hMSH2 (n = 1), or both (n = 1), whereas four MIN+/mutation- cases demonstrated normal expression for both. None of the MIN-/mutation- cases (n = 9) demonstrated an altered expression pattern for either protein. These data suggest that examination of protein expression by immunohistochemistry may be a rapid method for prescreening tumors for mutations in the MMR genes.

Human cytomegalovirus US3 impairs transport and maturation of major histocompatibility complex class I heavy chains

Abstract: The human cytomegalovirus (HCMV) early glycoprotein products of the US11 and US2 open reading frames cause increased turnover of major histocompatibility complex (MHC) class I heavy chains. Since US2 is homologous to another HCMV gene (US3), we hypothesized that the US3 gene product also may affect MHC class I expression. In cells constitutively expressing the HCMV US3 gene, MHC class I heavy chains formed a stable complex with beta 2-microglobulin. However, maturation of the N-linked glycan of MHC class I heavy chains was impaired in US3+ cells. The glycoprotein product of US3 (gpUS3) occurs mostly in a high-mannose form and coimmunoprecipitates with beta 2-microglobulin associated class I heavy chains. Mature class I molecules were detected at steady state on the surface of US3+ cells, as in control cells. Substantial perinuclear accumulation of heavy chains was observed in US3+ cells. The data suggest that gpUS3 impairs egress of MHC class I heavy chains from the endoplasmic reticulum.

Role of bone substitutes.

Abstract: Approximately 500 million years ago, the Paleozoic era heralded an evolutionary marvel : the skeleton. Unique to this evolutionary development was the capacity for regeneration : the physiologic renewal of embryologically derived tissue. Many of the cellular and molecular components for bone regeneration have been identified (bone morphogenetic proteins), and their therapeutic manipulation will become common clinical practice. Moreover, synthetic materials produced in the laboratory and novel bone derivatives will be used to exploit the skeleton's capacity to regenerate and repair. The concept of repair may be viewed as the restoration of form and function to deficient osseous tissue. Materials that provoke repair can be categorized broadly as bone substitutes. In this review, bone substitutes are grouped into 2 categories, polymers and ceramics, and each is subclassified as biodegradable or nonbiodegradable. Examples of these materials are provided as well as some of their liabilities and virtues.

Periprosthetic fractures of the femur. An analysis of 93 fractures.

Abstract: A retrospective review of 93 periprosthetic fractures and 102 periprosthetic fracture treatments showed that the type of prosthesis (cemented, ingrowth, Austin-Moore) and the presence of preexisting stress risers play a role in determining where the fractures occur. The site of fracture and the prefracture interface influence treatment of periprosthetic fractures. This study suggests that fractures associated with a loose interface, cemented or cementless, are best treated by removal of the prosthesis, reduction of the fracture, and insertion of a long stemmed prosthesis with additional fixation as needed. Treatment of a periprosthetic fracture associated with a stable prosthesis depends on the site of fracture. Fractures proximal to the tip of a fixed prosthesis usually can be treated nonoperatively or with limited internal fixation. Fractures at the tip of the prosthesis may be managed by revision or internal fixation, and fractures below the prosthesis can be managed operatively or nonoperatively.

Elevated alcohol consumption in null mutant mice lacking 5-HT1B serotonin receptors

Abstract: Substantial evidence links alcohol drinking and serotonin (5-HT) functioning in animals1. Lowered central 5-HT neurotransmission has been found in a subgroup of alcoholics, possibly those with more aggressive, assaultive tendencies2. Several rodent studies3 have also suggested that intact 5-HT systems are important determinants of sensitivity and/or tolerance to ethanol-induced ataxia and hypothermia. Null mutant mice lacking the 5-HT1B receptor gene (5-HT1B–/–) have been developed4 that display enhanced aggression5 and altered 5-HT release in slice preparations from some, but not all, brain areas6. We characterized these mice for sensitivity to several effects of ethanol. Mutant mice drank twice as much ethanol as wild-type mice, and voluntarily ingested solutions containing up to 20% ethanol in water. Their intake of food and water, and of sucrose, saccharin and quinine solutions, was normal. Mutants were less sensitive than wild-types on a test of ethanol-induced ataxia and, with repeated drug administration, tended to develop tolerance more slowly. In tests of ethanol withdrawal and metabolism, mutants and wild-type mice showed equivalent responses. Our results suggest that the 5-HT1B receptor participates in the regulation of ethanol drinking, and demonstrate that serotonergic manipulations lead to reduced responsiveness to certain ataxic effects of ethanol without affecting dependence.