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Thomas M Daly

Publications and source records attributed to Thomas M Daly.

17 recordsLinked to original sources
PubMed2006

Structure of the MTIP-MyoA complex, a key component of the malaria parasite invasion motor.

The causative agents of malaria have developed a sophisticated machinery for entering multiple cell types in the human and insect hosts. In this machinery, a critical interaction occurs between the unusual myosin motor MyoA and the MyoA-tail Interacting Protein (MTIP). Here we present one crystal structure that shows three different conformations of Plasmodium MTIP, one of these in complex with the MyoA-tail, which reveal major conformational changes in the C-terminal domain of MTIP upon binding the MyoA-tail helix, thereby creating several hydrophobic pockets in MTIP that are the recipients of key hydrophobic side chains of MyoA. Because we also show that the MyoA helix is able to block parasite growth, this provides avenues for designing antimalarials.

Amino Acid Sequence↗
PubMed2005

Precision profiling and components of variability analysis for Affymetrix microarray assays run in a clinical context.

Although gene expression profiling using microarray technology is widely used in research environments, adoption of microarray testing in clinical laboratories is currently limited. In an attempt to determine how such assays would perform in a clinical laboratory, we evaluated the analytical variability of Affymetrix microarray probesets using two generations of human Affymetrix chips (U95Av2 and U133A). The study was designed to mimic potential clinical applications by using multiple operators, machines, and reagent lots, and by performing analyses throughout a period of several months. A mixed model analysis was used to evaluate the relative contributions of multiple factors to overall variability, including operator, instrument, run, cRNA/cDNA synthesis, and changes in reagent lots. Under these conditions, the average probeset coefficient of variation (CV) was relatively low for present probesets on both generations of chips (mean coefficient of variation, 21.9% and 27.2% for U95Av2 and U133A chips, respectively). The largest contribution to overall variation was chip-to-chip (residual) variability, which was responsible for between 40 to 60% of the total variability observed. Changes in individual reagent lots and instrumentation contributed very little to the overall variability. We conclude that the approach demonstrated here could be applied to clinical validation of Affymetrix-based assays and that the analytical precision of this technique is sufficient to answer many biological questions.

DNA, Complementary↗
PubMed2005

Alteration in host cell tropism limits the efficacy of immunization with a surface protein of malaria merozoites.

Immunization with Plasmodium yoelii merozoite surface protein-8 (PyMSP-8) has been shown to protect mice against lethal P. yoelii 17XL malaria. Here we demonstrate that PyMSP-8-specific antibodies preferentially suppress P. yoelii 17XL growth in mature erythrocytes compared to growth in reticulocytes and do not suppress the growth of nonlethal P. yoelii 17X, a parasite that primarily replicates in reticulocytes. The protection against normocyte-associated P. yoelii malaria parasites is mediated by antibodies that recognize conformational epitopes of PyMSP-8 that are nonpolymorphic. We examined changes in gene expression in reticulocyte-restricted P. yoelii 17XL parasites that escaped neutralization by PyMSP-8-specific antibodies using P. yoelii DNA microarrays. Of interest, Pymsp-8 gene expression decreased, while the expression of msp-1, msp-7, and several rhoptry protein genes increased. Breakthrough parasites also exhibited increases in the expression of a subset of yir and Pyst-a genes that are predicted to encode polymorphic antigens expressed on the surface of infected erythrocytes. These data suggest that changes in the expression of parasite proteins expressed on the merozoite surface, as well as the surface of infected erythrocytes, may alter host cell tropism and contribute to the ability of malaria parasites to evade merozoite-specific, neutralizing antibodies.

Animals↗
PubMed2004

Regulation by phosphorylation of Pho81p, a cyclin-dependent kinase inhibitor in Saccharomyces cerevisiae.

Cyclins, cyclin-dependent kinases (CDK) and their inhibitors play a critical role in many biological processes. In yeast, the ankyrin repeat protein Pho81p, by being an inhibitor of the Pho85p-Pho80p cyclin-dependent protein kinase complex, transcriptionally regulates the production of repressible acid phosphatase, encoded by the PHO5 gene. Recent studies in our laboratory showed that Pho81p is phosphorylated by the Pho80p-Pho85p CDK complex in vitro; and, to determine the significance of the phosphorylation, we used site-directed mutagenesis to alter the potential phosphorylation sites for this kinase complex. The resulting mutations were introduced into a yeast strain containing a deletion of PHO81 and the effect of the mutation on PHO5 expression was assayed. Results suggest that phosphorylation of particular residues within Pho81p is crucial for its activity as an inhibitor. Studies using a green fluorescent protein-Pho81p fusion and Western analysis indicate that the localization and half-life of the mutants are similar to wild-type Pho81 proteins. However, an in vivo binding assay indicates that the mutant Pho81p is deficient in binding to the Pho80p-Pho85p kinase complex. These findings support the observation that the mutant fails to inhibit kinase activity in low phosphate. These studies provide insight into the mechanism of regulation of CDK inhibitor activity.

Acid Phosphatase↗
PubMed2004

Overestimation of HDL-cholesterol using a homogeneous "direct" assay.

Direct HDL-cholesterol (HDLc) assays have several advantages over other assays that are more laborious and time-consuming. A recent College of American Pathologists (CAP) report indicates that at least 385 Beckman LX-20 analyzers use the selective detergent (homogeneous or direct) method for analyzing HDLc. There is no published evaluation of direct HDLc assays on the Beckman platform. Here we report our evaluation of a direct HDLc assay marketed by Beckman for the Beckman LX-20 analyzers. In general, the assay performed well; however, the total error was not within National Cholesterol Education Program (NCEP) guidelines. This was largely because of a significant positive bias that appeared to be due, at least in part, to triglycerides. This bias was worse at HDLc concentrations <40 mg/dL, which may make it difficult to identify a low HDLc concentration and consequently an increased risk for cardiovascular disease (CVD).

Autoanalysis↗
PubMed2004

Members of the merozoite surface protein 7 family with similar expression patterns differ in ability to protect against Plasmodium yoelii malaria.

Previously, we described the isolation of the Plasmodium yoelii sequence-related molecules P. yoelii MSP-7 (merozoite surface protein 7) and P. yoelii MSRP-2 (MSP-7-related protein 2) by their ability to interact with the amino-terminal end of P. yoelii MSP-1 in a yeast two-hybrid system. One of these molecules was the homologue of Plasmodium falciparum MSP-7, which was biochemically isolated as part of the shed MSP-1 complex. In the present study, with antibodies directed against recombinant proteins, immunoprecipitation analyses of the rodent system demonstrated that both P. yoelii MSP-7 and P. yoelii MSRP-2 could be isolated from parasite lysates and from parasite culture supernatants. Immunofluorescence studies colocalized P. yoelii MSP-7 and P. yoelii MSRP-2 with the amino-terminal portion of MSP-1 and with each other on the surface of schizonts. Immunization with P. yoelii MSRP-2 but not P. yoelii MSP-7 protected mice against a lethal infection with P. yoelii strain 17XL. These results establish that both P. yoelii MSP-7 and P. yoelii MSRP-2 are expressed on the surface of merozoites and released from the parasite and that P. yoelii MSRP-2 may be the target of a protective immune response.

Animals↗
PubMed2004

Overview of adeno-associated viral vectors.

The use of adeno-associated virus (AAV) as a gene transfer vector has been steadily increasing over the past several years. AAV vectors have been particularly useful for applications where sustained gene expression is required. Prolonged in vivo expression following AAV treatment has been seen in the liver (1,2), brain (3,4), skeletal muscle (5,6), lung (7,8), and hematopoietic stem cells (9,10) of animal models. Therapeutic benefit from AAV treatment has been shown in a number of preclinical models of disease, including animal models of coagulopathies (11,12), lysosomal storage diseases (13,14), vision defects (15,16), and amino acid disorders. Clinical trials using AAV for the treatment of hemophilia B have begun, and early reports from these trials have been promising. In this introductory chapter to AAV, we will provide a brief overview of the molecular biology of this virus, an overview of methods of vector production, and a brief summary of the use of alternate AAV serotypes. The following chapters will then focus on specific methods and techniques for AAV transduction of the organs listed previously.

Dependovirus↗
PubMed2004

AAV-mediated gene transfer to the liver.

The liver is a frequent target of gene-transfer experiments, because of its central role in many metabolic and synthetic pathways. For applications where prolonged expression of genes in the liver is required, adeno-associated virus (AAV) has proven to be an effective tool for in vivo gene transfer. High-level, persistent hepatic expression has been achieved in a number of experimental systems following a single treatment with AAV in murine and larger animal models. This prolonged expression is particularly useful for the treatment of genetic diseases such as the inborn errors of metabolism, where lifelong expression of the deficient enzyme may be required. Therapeutic benefits using AAV vectors have been demonstrated in animal models of amino acid disorders, lysosomal storage diseases, and coagulopathies (3-5), and Phase I clinical trials are proposed for the treatment of hemophilia B (6). Gene transfer to the murine liver using AAV is achieved by intravenous (iv) injection of recombinant virus, either via a peripheral or portal vein. The liver is the primary organ transduced following intravenous injection of AAV, although other tissues such as heart and lung may also take up virus to a lesser extent when peripheral injection sites are used (7). Portal-vein injection can reduce the amount of extra-hepatic transduction, and allows a larger dose of virus to be delivered to the liver. However, this technique requires surgical expertise, and can only be performed on adult mice.

Animals↗
PubMed2003

A co-ligand complex anchors Plasmodium falciparum merozoites to the erythrocyte invasion receptor band 3.

In Plasmodium falciparum malaria, erythrocyte invasion by circulating merozoites may occur via two distinct pathways involving either a sialic acid-dependent or -independent mechanism. Earlier, we identified two nonglycosylated exofacial regions of erythrocyte band 3 termed 5ABC and 6A as an important host receptor in the sialic acid-independent invasion pathway. 5ABC, a major segment of this receptor, interacts with the 42-kDa processing product of merozoite surface protein 1 (MSP1(42)) through its 19-kDa C-terminal domain. Here, we show that two regions of merozoite surface protein 9 (MSP9), also known as acidic basic repeat antigen, interact directly with 5ABC during erythrocyte invasion by P. falciparum. Native MSP9 as well as recombinant polypeptides derived from two regions of MSP9 (MSP9/Delta1 and MSP9/Delta2) interacted with both 5ABC and intact erythrocytes. Soluble 5ABC added to the assay mixture drastically diminished the binding of MSP9 to erythrocytes. Recombinant MSP9/Delta1 and MSP9/Delta2 present in the culture medium blocked P. falciparum reinvasion into erythrocytes in vitro. Native MSP9 and MSP1(42), the two ligands binding to the 5ABC receptor, existed as a stable complex. Our results establish a novel concept wherein the merozoite exploits a specific complex of co-ligands on its surface to target a single erythrocyte receptor during invasion. This new paradigm poses a new challenge in the development of a vaccine for blood stage malaria.

Animals↗
PubMed2003

Myosin A tail domain interacting protein (MTIP) localizes to the inner membrane complex of Plasmodium sporozoites.

Apicomplexan host cell invasion and gliding motility depend on the parasite's actomyosin system located beneath the plasma membrane of invasive stages. Myosin A (MyoA), a class XIV unconventional myosin, is the motor protein. A model has been proposed to explain how the actomyosin motor operates but little is known about the components, topology and connectivity of the motor complex. Using the MyoA neck and tail domain as bait in a yeast two-hybrid screen we identified MTIP, a novel 24 kDa protein that interacts with MyoA. Deletion analysis shows that the 15 amino-acid C-terminal tail domain of MyoA, rather than the neck domain, specifically interacts with MTIP. In Plasmodium sporozoites MTIP localizes to the inner membrane complex (IMC), where it is found clustered with MyoA. The data support a model for apicomplexan motility and invasion in which the MyoA motor protein is associated via its tail domain with MTIP, immobilizing it at the outer IMC membrane. The head domain of the immobilized MyoA moves actin filaments that, directly or via a bridging protein, connect to the cytoplasmic domain of a transmembrane protein of the TRAP family. The actin/TRAP complex is then redistributed by the stationary MyoA from the anterior to the posterior end of the zoite, leading to its forward movement on a substrate or to penetration of a host cell.

Actin Cytoskeleton↗
PubMed2003

Polydrug fatality involving metaxalone.

A 29-year old female with a history of depression was found dead in a hotel room. The death scene investigation found empty pill bottles and an empty liter bottle of wine. Metaxalone, a centrally acting muscle relaxant, along with citalopram, ethanol, and chlorpheniramine were identified in the postmortem samples and quantitated by gas chromatography-mass spectrometry. The concentration of metaxalone in femoral vein blood was 39 mg/L. The heart blood concentration was 54 mg/L. Femoral vein blood concentrations of citalopram and chlorpheniramine were 0.77 mg/L and 0.04 mg/L, respectively. Ethanol levels were 0.13 g/dL in vitreous and 0.08 g/dL in heart blood. Other tissue samples were also analyzed. The authors consider the metaxalone concentrations toxic and potentially fatal. The citalopram concentrations were lower than those reported in fatal cases for this drug alone. Death was ascribed to polydrug abuse/overdose with metaxalone a major contributor. This represents the first reported case to our knowledge in which a metaxalone overdose significantly contributed to death.

Adult↗
PubMed2002

Comparison of non-diglyceride- and diglyceride-based assays for pancreatic lipase activity.

Pancreatic lipase is produced and stored in pancreatic acinar cells, and is normally secreted into the duct system. In disorders that cause pancreatic damage, pancreatic lipase enters the circulation, and serum lipase activity becomes useful in the diagnosis and evaluation of pancreatitis. During the last decade, many lipase assays have used the Imamura method, in which a series of enzymatic reactions utilizing a diglyceride substrate leads to the formation of a colorimetrically detected product. Historically, this method has been prone to significant interference from various substances, including glycerol itself. In the light of these limitations, we evaluated an automated enzymatic rate assay that uses a non-diglyceride-based, pancreatic lipase-specific substrate. Precision, linearity, and potential interference were assessed, and when compared to the Imamura method, the non-diglyceride-based assay exhibited a slope of 0.475, y-intercept of 15.89, r-value of 0.9516, and S(y,x) of 12.96. Similar results were also observed when the two assays were compared using samples with markedly elevated creatinine levels. Between-day coefficients of variance (CVs) ranged from 5.0% to 5.5%, which compared well with the diglyceride-based method, and linearity spanned a range of 3-156 U/L. Evaluation of over 2,000 patient results collected during a 6-month period suggested that the manufacturer's upper reference limit of 51 U/L may be too conservative. No significant interference was identified with bilirubin, triglyceride, or glycerol itself, and significant interference from hemoglobin was observed only at concentrations of 116 mg/dL or greater. As a result, the non-diglyceride-based method provides an acceptable alternative for the routine laboratory measurement of lipase activity.

Clinical Laboratory Techniques↗
PubMed2002

Adeno-associated virus-mediated gene transfer to the neonatal brain.

For many metabolic diseases, early treatment is necessary to prevent irreversible developmental damage. This is particularly true for childhood diseases that affect the central nervous system (CNS). The development of effective techniques for gene transfer to the neonatal brain would provide a new set of therapeutic options for many of these disorders. Vectors based on adeno-associated virus (AAV) have shown promise as agents for neonatal CNS transduction. In preclinical animal models, a single treatment with AAV vectors at birth has been shown to produce persistent CNS expression of transduced genes into adulthood. Transduction of the neonatal brain has been accomplished by a variety of methods, including direct intraparenchymal injection, intraventricular infusion, and intravenous administration. Of these methods, intraparenchymal injection provides the highest levels of localized activity, while intraventricular infusion results in a more widespread distribution of activity when performed in the neonate. Here we describe a method for direct, intraparenchymal injection of AAV into the neonatal brain. This technique provides a method for investigators to evaluate the effects of in vivo expression of exogenous genes on the process of early brain development.

Adenoviridae↗
PubMed2002

A multigene family that interacts with the amino terminus of plasmodium MSP-1 identified using the yeast two-hybrid system.

Merozoite surface protein 1 (MSP-1) is a high-molecular-weight protein expressed on the surface of the malaria merozoite in a noncovalent complex with other protein molecules. MSP-1 undergoes a series of proteolytic processing events, but no precise biological role for the various proteolytic fragments of MSP-1 or for the additional proteins present in the complex is known. Through the use of the yeast two-hybrid system, we have isolated genes encoding proteins that interact with a region of the amino-terminal proteolytic fragment of MSP-1 from the mouse parasite Plasmodium yoelii. This analysis has led to the isolation of two sequence-related molecules, one of which is the P. yoelii homologue of MSP-7 originally described in Plasmodium falciparum. BLAST analysis of the P. falciparum database has revealed that there are six related protein molecules present in this species encoded near each other on chromosome 13. In P. falciparum, we designated these molecules MSRP-1 to -5. Analysis of the P. yoelii database indicates a similar chromosomal organization for the two genes in the mouse parasite species. The three P. falciparum sequences with the highest degree of homology to the P. yoelii sequences isolated in the two-hybrid screen have been characterized at the molecular level (MSRP-1 to -3). Expression analysis indicated that the mRNAs are expressed at various levels in the different asexual stages. Immunofluorescence studies colocalized the expression of the MSRP molecules and the amino-terminal portion of MSP-1 to the surfaces of trophozoites. In vitro binding experiments confirmed the interaction between MSRP-1, MSRP-2, and the amino-terminal region of P. falciparum MSP-1.

Amino Acid Sequence↗
PubMed2002

Evaluation of a no-pretreatment cyclosporin A assay on the Dade Behring dimension RxL clinical chemistry analyzer.

BACKGROUND: Monitoring whole-blood concentrations of cyclosporin A (CsA) is common practice in the management of solid organ and bone marrow transplant recipients. In a multicenter study we evaluated a new, direct (no pretreatment) CsA assay on the Dade Behring Dimension RxL system and compared results with those from the Abbott TDx CsA immunoassay and a HPLC method. METHODS: Whole-blood samples from heart (n = 111; 35 patients), liver (n = 201; 44 patients), kidney (n = 279; 65 patients), and miscellaneous organ (n = 77; 12 lung, 12 bone marrow, 5 kidney/pancreas, and 1 pancreas patient) recipients were obtained from patient populations of the participating institutions. Routine clinical monitoring of CsA was performed using either the TDx method or HPLC. RESULTS: The minimum detectable concentration of CsA averaged 9.4 microg/L, and the lower limit of quantification was 30 microg/L. The method was linear from 30 to 500 microg/L. Cross-reactivity with seven different CsA metabolites ranged from 0.0% to 5.7% for the Dimension RxL assay compared with 0.4-15.9% for the TDx assay. Total imprecision (CV) averaged 6.2%, and within-run imprecision averaged 4.9%. Passing-Bablok linear regression analyses of all samples from two sites yielded the following: RxL = 0.81 x TDx - 16.8; and RxL = 1.12 x HPLC - 1.7. CONCLUSIONS: The Dade Behring CsA assay for the random-access Dimension platform offers adequate performance characteristics for routine clinical use, does not require a manual pretreatment step, and demonstrates less cross-reactivity with CsA metabolites than another commonly used immunoassay.

Antibodies, Monoclonal↗