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Biomedical subjects

D M Miller

Publications and source records attributed to D M Miller.

At least 55 records · Page 3Linked to original sources
PubMed2000

Immunohistochemical and clinical evaluation of p53 in canine cutaneous mast cell tumors.

One hundred twenty-six cutaneous mast cell tumors obtained by excisional biopsy from 106 dogs were evaluated using immunohistochemical staining for the presence of p53 protein. A standard avidin-biotin immunohistochemical protocol was used incorporating a polyclonal antibody of rabbit origin (CM-1) as the primary antibody. Histopathologic grading of tumors was performed on hemotoxylin and eosin-stained samples. There was a significant difference in the percentage of cells staining positive for p53 for the histopathologic grades (P = 0.0005). Grade III tumors had a significantly greater p53 content than did grade I or II tumors (P < 0.05). Clinical data obtained retrospectively was available for 54 dogs. Tumor recurred in 19 of 54 (35.2%) dogs. Twenty-nine dogs died by the end of the study; 9 of 29 (31.0%) died of mast cell tumor disease. Histopathologic grade showed a significant negative association with survival time. Both clinical stage and histopathologic grade showed a significant negative association with time to recurrence. The percentage of cells staining positive for p53 did not significantly improve the forward analysis. Immunohistochemical detection of p53 did not appear useful in characterizing the clinical association between cutaneous mast cell tumor cellular features and survival time or time to tumor recurrence in dogs.

Animals↗
PubMed2000

Novel extraction of German cockroach (Dictyoptera: Blattellidae) fecal pellets enhances efficacy of spray formulation insecticides.

Methanol extracts of German cockroach, Blattella germanica (L.), fecal pellets have limited use as pheromone attractants in the urban environment because of their unpleasant color and odor. To eliminate these characteristics, a novel aqueous extract of German cockroach feces was formulated. Fetal material was extracted with methylene chloride and mixed with water. The aqueous phase of the extract was colorless and relatively odorless compared with methanol fecal extracts. Aqueous extract was bioassayed and compared with methanol extracts for aggregation activity. The efficacy of chlorpyrifos and boric acid formulations was tested with and without the addition of fecal extracts. In tests with chlorpyrifos, the addition of the aqueous extract produced significantly greater mortality in young nymphs than either chlorpyrifos alone or chlorpyrifos + methanol extract. There were no differences in mortality between the 2 extract + boric acid treatments during the test period, and both enhanced mortality.

Animals↗
PubMed2000

Use of fecal extract trails to enhance trap catch in German cockroach (Dictyoptera: Blattellidae) monitoring stations.

An aqueous extract of German cockroach, Blattella germanica (L.), fecal material was evaluated for inducing trail-following behavior in German cockroaches. In arena tests the fecal extract was found to stimulate trail following in 74% of adult male cockroaches. Significantly fewer cockroaches (22%) followed water-treated (control) trails. Residual activity of the fecal extract trails was evaluated by bioassay after the trails had been stored in the refrigerator or in the open air. Although trails stored in the refrigerator showed no decline in activity after 14 d, those stored in the open air declined significantly after 3 d, inducing only 40% of adult male cockroaches to follow the trail. After 7 d the activity of trails stored in the open air was further reduced to 23%. The ability of fecal-extract trails to influence trap catch in monitoring stations was determined by bioassay. Paper trails treated with fecal extract or water were positioned between cockroach harborages and monitoring stations inside 122-cm2 arenas. The presence of the fecal extract-treated trails significantly enhanced trap catch. Mean catch in the traps with fecal extract trails was 28 cockroaches compared with a mean of 11 cockroaches in the control traps. The trap catch ratios of adults to nymphs in the treated and control treatments were not significantly different.

Animals↗
PubMed2000

Trail-following behavior in the German cockroach (Dictyoptera: Blattellidae).

The trail-following behavior of the German cockroach, Blattella germanica (L.), was evaluated by comparing the distance between cockroach movement paths and preapplied "trails" of fecal extract. For each cockroach group tested (adult males, females, gravid females, and late instars), the mean perpendicular distance of the cockroach from the trail was significantly less than the distance from a control trail. The results indicated that the German cockroaches did exhibit trail-following behavior. Trail- following accuracy varied among the cockroach groups. The mean distance from the fecal trail ranged from 18.45 to 110.05 cm with adult males < or = adult females < or = late instars < gravid females. Very dilute fecal extract could still induce trail-following behavior in adult male cockroaches. A 5.6% concentration of fecal extract in methanol was able to induce trail-following behavior in 50% of the cockroaches. Although German cockroaches have demonstrated trail-following behavior, fecal trails are still not thought to be actively deposited. Rather, the passive distribution of fecal material within the home range results in the accumulation of trails along frequently traveled routes (i.e., between resources and the cockroach harborage).

Animals↗
PubMed1999

UNC-4/UNC-37-dependent repression of motor neuron-specific genes controls synaptic choice in Caenorhabditis elegans.

The UNC-4 homeoprotein and the Groucho-like corepressor UNC-37 specify synaptic choice in the Caenorhabditis elegans motor neuron circuit. In unc-4 mutants, VA motor neurons are miswired with inputs from interneurons normally reserved for their lineal sisters, the VB motor neurons. Here we show that UNC-4 and UNC-37 function together in VA motor neurons to repress VB-specific genes and that this activity depends on physical contact between UNC-37 and a conserved Engrailed-like repressor domain (eh1) in UNC-4. Missense mutations in the UNC-4 eh1 domain disrupt interactions between UNC-4 and UNC-37 and result in the loss of UNC-4-dependent repressor activity in vivo. A compensatory amino acid substitution in UNC-37 suppresses specific unc-4 alleles by restoring physical interactions with UNC-4 as well as UNC-4-dependent repression of VB-specific genes. We propose that repression of VB-specific genes by UNC-4 and UNC-37 is necessary for the creation of wild-type inputs to VA motor neurons. The existence of mammalian homologs of UNC-4 and UNC-37 indicates that a similar mechanism could regulate synaptic choice in the vertebrate spinal cord.

Amino Acid Sequence↗
PubMed1999

Novel mechanism of inhibition of cytomegalovirus by the experimental immunosuppressive agent leflunomide.

BACKGROUND: Despite progress in antiviral chemotherapy, cytomegalovirus (CMV) remains a major cause of morbidity and mortality among pharmacologically immunosuppressed organ transplant recipients, frequently engaging the clinician in a struggle to balance graft preservation with control of CMV disease. Leflunomide, an inhibitor of protein kinase activity and pyrimidine synthesis, is an experimental immunosuppressive agent effective against acute and chronic allograft rejection in animal models. Because a number of CMV proteins are known to be phosphorylated, we tested the hypothesis that this agent might exert inhibitory activity against CMV. METHODS AND RESULTS: Plaque assays demonstrated dramatic dose-dependent attenuation of production of multiple clinical CMV isolates in leflunomide-treated human fibroblasts and endothelial cells, common targets for CMV infection in vivo. As shown by Northern blot analysis and immunohistochemical staining, leflunomide neither interferes with transcription of immediate early or late viral genes, nor with expression of corresponding proteins. CMV-specific DNA dot blots and biochemical enzyme assays indicated that, in contrast to currently approved anti-CMV drugs, leflunomide exerts no inhibitory effect on the accumulation of viral DNA in infected cells, or on viral DNA polymerase activity. Rather, as visualized by transmission electron microscopy, this agent appears to act at a late stage in virion assembly by preventing tegument acquisition by viral nucleocapsids. Finally we have demonstrated equivalent inhibitory activity of leflunomide against multi-drug-resistant CMV isolates. CONCLUSIONS: These findings imply that leflunomide, an effective immunosuppressive agent, shows potential to concurrently attenuate a major complication of immunosuppression, CMV disease, by a novel mechanism of antiviral activity.

Antiviral Agents↗
PubMed1999

Antiproliferative activity of G-rich oligonucleotides correlates with protein binding.

Oligonucleotides have been extensively studied as antisense or antigene agents that can potentially modulate the expression of specific genes. These strategies rely on sequence-specific hybridization of the oligonucleotide to mRNA or genomic DNA. Recently, it has become clear that oligonucleotides often have biological activities that cannot be attributed to their sequence-specific interactions with nucleic acids. Here we describe a series of guanosine-rich phosphodiester oligodeoxynucleotides that strongly inhibit proliferation in a number of human tumor cell lines. The presence of G-quartets in the active oligonucleotides is demonstrated using an UV melting technique. We show that G-rich oligonucleotides bind to a specific cellular protein and that the biological activity of the oligonucleotides correlates with binding to this protein. The G-rich oligonucleotide-binding protein was detected in both nuclear and cytoplasmic extracts and in proteins derived from the plasma membrane of cells. We present strong evidence that this protein is nucleolin, a multifunctional phosphoprotein whose levels are related to the rate of cell proliferation. Our results indicate that binding of G-rich oligonucleotides to nucleolin may be responsible for their non-sequence-specific effects. Furthermore, these oligonucleotides represent a new class of potentially therapeutic agents with a novel mechanism of action.

Base Sequence↗
PubMed1999

Human cytomegalovirus inhibits IFN-alpha-stimulated antiviral and immunoregulatory responses by blocking multiple levels of IFN-alpha signal transduction.

The type I IFNs represent a primordial, tightly regulated defense system against acute viral infection. IFN-alpha confers resistance to viral infection by activating a conserved signal transduction pathway that up-regulates direct antiviral effectors and induces immunomodulatory activities. Given the critical role of IFN-alpha in anti-human cytomegalovirus (HCMV) immunity and the profound ability of HCMV to escape the host immune response, we hypothesized that HCMV blocks IFN-alpha-stimulated responses by disrupting multiple levels of the IFN-alpha signal transduction pathway. We demonstrate that HCMV inhibits IFN-alpha-stimulated MHC class I, IFN regulatory factor-1, MxA and 2',5-oligoadenylate synthetase gene expression, transcription factor activation, and signaling in infected fibroblasts and endothelial cells by decreasing the expression of Janus kinase 1 and p48, two essential components of the IFN-alpha signal transduction pathway. This investigation is the first to report inhibition of type I IFN signaling by a herpesvirus. We propose that this novel immune escape mechanism is a major means by which HCMV is capable of escaping host immunity and establishing persistence.

Antiviral Agents↗
PubMed1999

The integral divalent cation within the intermolecular purine*purine. pyrimidine structure: a variable determinant of the potential for and characteristics of the triple helical association.

In vitro assembly of an intermolecular purine*purine.pyrimidine triple helix requires the presence of a divalent cation. The relationships between cation coordination and triplex assembly were investigated, and we have obtained new evidence for at least three functionally distinct potential modes of divalent cation coordination. (i) The positive influence of the divalent cation on the affinity of the third strand for its specific target correlates with affinity of the cation for coordination to phosphate. (ii) Once assembled, the integrity of the triple helical structure remains dependent upon its divalent cation component. A mode of heterocyclic coordination/chelation is favorable to triplex formation by decreasing the relative tendency for efflux of integral cations from within the triple helical structure. (iii) There is also a detrimental mode of base coordination through which a divalent cation may actively antagonize triplex assembly, even in the presence of other supportive divalent cations. These results demonstrate the considerable impact of the cationic component, and suggest ways in which the triple helical association might be positively or negatively modulated.

Base Pairing↗
PubMed1999

Viral effects on antigen processing.

Viruses have evolved numerous mechanisms that modulate MHC-mediated antigen presentation, which in turn protect infected cells from T-lymphocyte-mediated immunosurveillance. Recent studies of previously identified viral immunomodulatory proteins reveal the allelic specificity of these proteins, their ability to function in xenogeneic systems and the difficulty in translating in vitro data to in vivo models; moreover, new mechanisms of viral modulation of MHC expression have emerged.

Animals↗
PubMed1999

Viral inhibition of interferon signal transduction.

The type I and II interferons (IFNs) are potent stimulators of antigen processing and presentation and are essential in antiviral immunity. IFNs upregulate the transcription of major histocompatibility complex (MHC) class I and II molecules, associated antigen-processing proteins, and induce the production of direct antiviral effector molecules such as 2',5'-oligoadenylate synthetase, double-stranded-RNA-dependent protein kinase and Mx proteins. It is increasingly evident that viruses have evolved mechanisms to globally inhibit the actions of IFNs through disruption of their signal transduction pathways. Herein, we review the ability and novel mechanisms of several diverse viruses to inhibit IFN-induced JAK/STAT signal transduction.

Humans↗
PubMed1999

Inhibition of cytomegalovirus in vitro and in vivo by the experimental immunosuppressive agent leflunomide.

Despite progress in antiviral chemotherapy, cytomegalovirus (CMV) remains a major cause of morbidity and mortality among pharmacologically immunosuppressed transplant recipients, frequently engaging the clinician in a struggle to balance graft preservation with control of CMV disease. Leflunomide, an inhibitor of protein kinase activity and pyrimidine synthesis, is an experimental immunosuppressive agent effective against acute and chronic rejection in animal models. Herein we summarize our recent studies demonstrating that leflunomide inhibits the production of multiple clinical CMV isolates (including multi-drug-resistant virus) in both human fibroblasts and endothelial cells. In contrast to all other anti-CMV drugs currently in use, leflunomide does not inhibit viral DNA synthesis, but rather appears to interfere with virion assembly. Finally, preliminary studies in a rat model suggest that this agent reduces viral load in vivo. These findings imply that leflunomide, an effective immunosuppressive agent, shows potential to concurrently attenuate a major complication of immunosuppression, CMV disease, by a novel mechanism of antiviral activity.

Animals↗
PubMed1999

Recent developments in the assessment of quality of life in multiple sclerosis (MS).

Multiple sclerosis (MS) and its treatment have broad-ranging effects on quality of life. This article reviews recent efforts to assess the impact of MS on activities of daily living (ADLs) and health-related quality of life (HRQL), and describes the development of the Multiple Sclerosis Quality of Life Inventory (MSQLI). The MSQLI is a modular MS-specific HRQL instrument consisting of a widely-used generic measure, the Health Status Questionnaire (SF-36), supplemented by nine symptom-specific measures (covering fatigue, pain, bladder function, bowel function, emotional status, perceived cognitive function, visual function, sexual satisfaction, and social relationships). Content validation consisted of evaluating its adherence to a conceptual model of the impact of MS, and review by MS specialists (neurologists and allied health professionals), HRQL experts, patients, and caregivers. The reliability and construct validity of the MSQLI were rigorously evaluated in a field test with 300 North American patients (198 female, 102 male) with definite MS (Poser criteria) and a broad range of physical impairment (EDSS=0. 0-8.5). This article concludes by comparing the MSQLI with two other MS-specific HRQL measures (MS Quality of Life-54 (QOL-54) and Functional Assessment of Multiple Sclerosis (FAMS)) and discussing key issues to consider in selecting an HRQL instrument for a collaborative database.

Activities of Daily Living↗
PubMed1999

Two-color GFP expression system for C. elegans.

We describe the use of modified versions of the Aequora victoria green fluorescent protein (GFP) to simultaneously follow the expression and distribution of two different proteins in the nematode, Caenorhabditis elegans. A cyan-colored GFP derivative, designated CFP, contains amino acid (aa) substitutions Y66W, N146I, M153T and V163A relative to the original GFP sequence and is similar to the previously reported "W7" form. A yellow-shifted GFP derivative, designated YFP, contains aa substitutions S65G, V68A, S72A and T203Y and is similar to the previously described "I0C" variant. Coding regions for CFP and YFP were constructed in the context of a high-activity C. elegans expression system. Previously characterized promoters and localization signals have been used to express CFP and YFP in C. elegans. Filter sets designed to distinguish YFP and CFP fluorescence spectra allowed visualization of the two distinct forms of GFP in neurons and in muscle cells. A series of expression vectors carrying CFP and YFP have been constructed and are being made available to the scientific community.

Amino Acid Substitution↗
PubMed1998

Human cytomegalovirus inhibits major histocompatibility complex class II expression by disruption of the Jak/Stat pathway.

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that is able to persist for decades in its host. HCMV has evolved protean countermeasures for anti-HCMV cellular immunity that facilitate establishment of persistence. Recently it has been shown that HCMV inhibits interferon gamma (IFN-gamma)-stimulated MHC class II expression, but the mechanism for this effect is unknown. IFN-gamma signal transduction (Jak/Stat pathway) and class II transactivator (CIITA) are required components for IFN-gamma-stimulated MHC class II expression. In this study, we demonstrate that both a clinical isolate and a laboratory strain of HCMV inhibit inducible MHC class II expression at the cell surface and at RNA level in human endothelial cells and fibroblasts. Moreover, reverse transcriptase polymerase chain reaction and Northern blot analyses demonstrate that neither CIITA nor interferon regulatory factor 1 are upregulated in infected cells. Electrophoretic mobility shift assays reveal a defect in IFN-gamma signal transduction, which was shown by immunoprecipitation to be associated with a striking decrease in Janus kinase 1 (Jak1) levels. Proteasome inhibitor studies with carboxybenzyl-leucyl-leucyl-leucine vinyl sulfone suggest an HCMV-associated enhancement of Jak1 protein degradation. This is the first report of a mechanism for the HCMV-mediated disruption of inducible MHC class II expression and a direct virus-associated alteration in Janus kinase levels. These findings are yet another example of the diverse mechanisms by which HCMV avoids immunosurveillance and establishes persistence.

Cells, Cultured↗
PubMed1998

Inhibition of transcription of the human c-myc protooncogene by intermolecular triplex.

Triplex-forming oligonucleotides (TFOs) have been shown to inhibit both transcription in vitro and the expression of target genes in cell culture by binding to polypurine/polypyrimidine sequences in several human gene promoters. The c-myc protooncogene is overexpressed in a variety of human cancers and appears to play an important role in the proliferation of these cells. In an attempt to assay the ability of triplex-forming oligonucleotides to inhibit expression of a target gene in vivo, we have developed a cellular system involving transfection of a c-myc promoter-driven luciferase reporter plasmid with triplex-forming oligonucleotides targeted to the human c-myc protooncogene. To increase the stability of the TFO, we have used modified phosphorothioate oligonucleotides. Triplex formation with a modified phosphorothioate oligonucleotide occurs with approximately equal binding affinity as that seen using a phosphodiester oligonucleotide. Phosphorothioate-modified TFOs targeted to c-myc inhibit transcription of the c-myc promoter in HeLa cells as demonstrated by a decrease in luciferase expression from a luciferase reporter gene construct. These results suggests that triplex formation may represent a gene-specific means of inhibiting specific protooncogene expression.

Base Sequence↗
PubMed1998

A novel triplex-forming oligonucleotide targeted to human cyclin D1 (bcl-1, proto-oncogene) promoter inhibits transcription in HeLa cells.

The cyclin D1/bcl-1 proto-oncogene is one of a series of genes encoding proteins which regulate the cell cycle and are involved in the multistep process of tumorigenesis. Translocation of the cyclin D1 proto-oncogene is a common event in B cell lymphoma, and cyclin D1 amplification occurs in breast, esophageal, hepatocellular, and head/neck carcinomas. The human cyclin D1 proto-oncogene promoter contains an 18-base pair purine-pyrimidine rich motif with three C.G interruptions. This motif is a potential target for purine.purine. pyrimidine triplex formation. We have designed a G-rich antiparallel triplex forming oligonucleotide (TFO) targeted to this region. Electrophoretic mobility shift analysis (EMSA) shows that this purine-pyrimidine rich motif is a binding site for the transcription factor Sp1 and that triplex formation by the target sequence prevents the binding of recombinant Sp1. The exact location of triplex formation was confirmed by DNase I footprinting. In an attempt to increase stability, we have used modified phosphorothioate oligonucleotides for cell culture experiments. Triplex formation by the cyclin D1 targeted phosphorothioate oligonucleotide occurs with a binding affinity approximately equal to that of phosphodiester oligonucleotides. This phosphorothioate modified TFO targeted to cyclin D1 also inhibits transcription of the cyclin D1 promoter in HeLa cells, as demonstrated by a decrease in luciferase expression from a stably integrated human cyclin D1 promoter driven luciferase construct. This suggests that triplex formation may represent a gene specific means of inhibiting cyclin D1 expression.

Base Sequence↗