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K J Ryan

Publications and source records attributed to K J Ryan.

At least 19 recordsLinked to original sources
PubMed2000

Binding of PurH to a muscle-specific splicing enhancer functionally correlates with exon inclusion in vivo.

Regulated alternative splicing of avian cardiac troponin T (cTNT) pre-mRNA requires multiple intronic elements called muscle-specific splicing enhancers (MSEs) that flank the alternative exon 5 and promote muscle-specific exon inclusion. To understand the function of the MSEs in muscle-specific splicing, we sought to identify trans-acting factors that bind to these elements. MSE3, which is located 66-81 nucleotides downstream of exon 5, assembles a complex that is both sequence- and muscle-specific. Purification and characterization of the MSE3 complex identified one component as 5-aminoimidazole-4-carboxamide ribonucleotideformyltransferase/IMP cyclohydrolase (PurH), an enzyme involved in de novo purine synthesis. Recombinant human PurH protein directly binds MSE3 RNA and PurH is the primary determinant of sequence-specific binding in the native complex. Furthermore, we show a direct correlation between the in vitro binding affinity of both the MSE3 complex and recombinant PurH with functional activation of exon inclusion in vivo. Together, these results strongly suggest that PurH performs a second function as a component of a complex that regulates MSE3-dependent exon inclusion.

Alternative Splicing↗
PubMed2000

The nuclear pore complex: a protein machine bridging the nucleus and cytoplasm.

Compositional analysis of nuclear pore complexes (NPCs) is nearing completion, and efforts are now focused on understanding how these protein machines work. Recent analysis of soluble transport factor interactions with NPC proteins reveals distinct and overlapping pathways for movement between the nucleus and cytoplasm. New fluorescence- and microscopy-based strategies have been used to monitor the pathway of NPC assembly and to reveal the dynamics of the NPC during transport.

Animals↗
PubMed2000

Assembly and preferential localization of Nup116p on the cytoplasmic face of the nuclear pore complex by interaction with Nup82p.

The yeast Saccharomyces cerevisiae nucleoporin Nup116p serves as a docking site for both nuclear import and export factors. However, the mechanism for assembling Nup116p into the nuclear pore complex (NPC) has not been resolved. By conducting a two-hybrid screen with the carboxy (C)-terminal Nup116p region as bait, we identified Nup82p. The predicted coiled-coil region of Nup82p was not required for Nup116p interaction, making the binding requirements distinct from those for the Nsp1p-Nup82p-Nup159p subcomplex (N. Belgareh, C. Snay-Hodge, F. Pasteau, S. Dagher, C. N. Cole, and V. Doye, Mol. Biol. Cell 9:3475-3492, 1998). Immunoprecipitation experiments using yeast cell lysates resulted in the coisolation of a Nup116p-Nup82p subcomplex. Although the absence of Nup116p had no effect on the NPC localization of Nup82p, overexpression of C-terminal Nup116p in a nup116 null mutant resulted in Nup82p mislocalization. Moreover, NPC localization of Nup116p was specifically diminished in a nup82-Delta108 mutant after growth at 37 degrees C. Immunoelectron microscopy analysis showed Nup116p was localized on both the cytoplasmic and nuclear NPC faces. Its distribution was asymmetric with the majority at the cytoplasmic face. Taken together, these results suggest that Nup82p and Nup116p interact at the cytoplasmic NPC face, with nucleoplasmic Nup116p localization utilizing novel binding partners.

Cell Nucleus↗
PubMed1999

Research misconduct in clinical research--the American experience and response.

Research misconduct in the United States has occurred sporadically since 1961 in the laboratories of some of our most distinguished scientists. In view of the enormous number of research grants funded, cases of this kind are relatively uncommon, but have none the less attracted governmental supervision and calls for reform. The scientific community, universities and government have addressed the issue in various ways and changes have been proposed and some actually instituted. In view of human nature, no one seriously believes that dishonesty in research can be prevented to any greater extent than in any other human activity. However, some practices may discourage and mitigate such occurrences. These include: education in sound laboratory research practices, a fair distribution of authorship assignment, and adequate supervision of research personnel including appropriate reviewing of primary data. Other measures which might be considered include: an adequate check of the credentials of all new personnel, audits for clinical research, especially for those involving significant numbers of patients and multiple institutions, and the introduction of quality control concepts into research procedures. The hope is that the senior individual scientist responsible for the quality and integrity of the research will institute such measures as needed, and that institutional and government supervision will not interfere with the creative process.

Humans↗
PubMed1999

Experimental determination of section sensitivity profiles and image noise in electron beam computed tomography.

To determine the effect of continuous-volume scanning (CVS) on z-axis resolution, section sensitivity profiles were measured on an electron beam computed tomography (CT) scanner and compared with those obtained using the step-volume scanning (SVS) mode. A steel bead was imaged using different scan parameters, and the mean CT number over the bead was plotted against the z-axis position to determine section sensitivity profiles. From these profiles, full width at half maximum (FWHM), full width at tenth maximum (FWTM), and full width at tenth area (FWTA) were calculated. A uniform water phantom was imaged to measure noise. To determine the visual significance of changes in the section sensitivity profile, a section thickness and contiguity phantom was imaged. All section sensitivity profiles measured had an FWHM value within 0.5 mm of the nominal scan width. The FWTM and FWTA values increased with the CVS mode compared with the SVS mode. This broadening of the section sensitivity profiles was most significant with larger collimator widths. However, use of smaller collimator widths increased image noise. When all other parameters remained constant, increasing the exposure time to reduce image noise did not affect the section sensitivity profile. The CVS mode produced wider section sensitivity profiles than the SVS mode. This effect was minimized when the smallest collimator width was used, but at the expense of increased image noise.

Electrons↗
PubMed1999

Research integrity.

Explore the source record for details and available documents.

Biomedical Research↗
PubMed1998

Telemicrobiology: feasibility study.

BACKGROUND: Rural hospitals generally lack staffing with infectious disease specialists or pathologists. Without on-site pathologists, the range of microbiology services offered by clinical laboratories may be limited as well. OBJECTIVE: To study the feasibility of using static-image telepathology to evaluate Gram stains of microbiologic preparations. MATERIALS AND METHODS: In this retrospective feasibility study, three pathologists evaluated Gram stains of slides from 50 cases by two viewing modalities: static-image telepathology and conventional light microscopy. Digital video images of slides were captured at two magnifications (using 40x and 100x objective lenses) at 1024 x 768 x 24-bit color and transmitted over standard telephone lines at 14,400 kbps. Pathology reports and culture results served as "truth diagnoses." Categories of interpretations were correct, minor discrepancy, or major discrepancy with regard to the implications for patient care. RESULTS: The diagnostic accuracy of video image readings and conventional light microscopy readings were nearly identical, with no statistically significant differences in the performances of specialty and nonspecialty pathologists (P > 0.05). The mean accuracies of readings of the video images and light microscopy images were 95.3% and 95.4%, respectively. Taking into account the time required by a referring pathologist to capture video digital images, telemicrobiology was somewhat less efficient than conventional light microscopy. CONCLUSIONS: Pathologists can accurately evaluate digital video images of preselected fields on Gram-stained slides. In clinical practice, however, a limiting factor may be the availability of local personnel qualified to select the microscopic fields for evaluation by telepathologists. The adequacy of the video images suggests that telepathology may also be used for remote supervision of quality assurance programs in microbiology laboratories, as well as for remote proficiency training of laboratory personnel.

Arizona↗
PubMed1998

1,2,4-Benzotriazine 1,4-dioxides. An important class of hypoxic cytotoxins with antitumor activity.

Tirapazamine (1,2,4-benzotriazin-3-amine 1,4-dioxide, SR 4233, WIN 59075) is the lead compound representing this class of anticancer drugs. It is also the first compound to be introduced in the clinic as a pure bioreductive cytotoxic agent. Tirapazamine represents a completely novel approach to the treatment of solid tumors and has generated considerable interest, with research being carried out on all aspects of the its anticancer activity. Phase III trials of tirapazamine in combination with cisplatin (cDDP) have recently been concluded, and phase II trials of triapazamine in combination with irradiation are presently being performed. We developed a drug discovery program into this class of compounds designed to produce derivatives with improved in vivo activity against solid tumors. Based on the hypothesis that these compounds require bioreductive activation for antitumor activity, the research was primarily directed at producing analogues with greater electron affinity and improved aqueous solubility. The in vitro and in vivo data for a variety of structural analogues clearly show that 1,2,4-benzotriazine 1,4-dioxides have considerable potential as anticancer agents. When their activity is compared directly with the activity observed for triapazamine, the most promising series of analogues appears to be the 3-alkyl-substituted derivatives, especially the 3-ethyl- and 3-(2'-methoxyethyl)-derivatives, SR 4895 and SR 4941 respectively.

Animals↗
PubMed1996

Muscle-specific splicing enhancers regulate inclusion of the cardiac troponin T alternative exon in embryonic skeletal muscle.

The alternative exon 5 of the striated muscle-specific cardiac troponin T (cTNT) gene is included in mRNA from embryonic skeletal and cardiac muscle and excluded in mRNA from the adult. The embryonic splicing pattern is reproduced in primary skeletal muscle cultures for both the endogenous gene and transiently transfected minigenes, whereas in nonmuscle cell lines, minigenes express a default exon skipping pattern. Using this experimental system, we previously showed that a purine-rich splicing enhancer in the alternative exon functions as a constitutive splicing element but not as a target for factors regulating cell-specific splicing. In this study, we identify four intron elements, one located upstream,and three located downstream of the alternative exon, which act in a positive manner to mediate the embryonic splicing pattern of exon inclusion. Synergistic interactions between at least three of the four elements are necessary and sufficient to regulate splicing of a heterologous alternative exon and heterologous splice sites. Mutations in these elements prevent activation of exon inclusion in muscle cells but do not affect the default level of exon inclusion in nonmuscle cells. Therefore, these elements function as muscle-specific splicing enhancers (MSEs) and are the first muscle-specific positive-acting splicing elements to be described. One MSE located downstream from the alternative exon is conserved in the rat and chicken cTNT genes. A related sequence is found in a third muscle-specific gene, that encoding skeletal troponin T, downstream from an alternative exon with a developmental pattern of alternative splicing similar to that of rat and chicken cTNT. Therefore, the MSEs identified in the cTNT gene may play a role in developmentally regulated alternative splicing in a number of different genes.

Alternative Splicing↗
PubMed1996

The pharmacokinetics, bioavailability and biodistribution in mice of a rationally designed 2-nitroimidazole hypoxia probe SR-4554.

N-(2-Hydroxy-3,3,3-trifluoropropyl)-2-(2-nitro-1-imidazolyl) acetamide (SR-4554) is a fluorinated 2-nitroimidazole which has been rationally designed as a non-invasive probe for tumor hypoxia. The key selection criteria for this molecule were low central nervous system penetration and toxicity, high metabolic stability other than nitroreduction, good tumor uptake and high sensitivity for detection by magnetic resonance spectroscopy. As part of the pre-clinical development strategy, pharmacokinetic, bioavailability and biodistribution studies were performed in mice. Pharmacokinetic studies in mice demonstrated that SR-4554 was rapidly absorbed into plasma following i.p. administration and eliminated with a half-life of 42 min, similar to other 2-nitroimidazoles. By comparing the areas under the concentration-time curve (AUC), the tumor exposure towards SR-4554 was on average 84% of the value obtained for the plasma exposure. SR-4554 penetrated tumor tissue extremely well but, in contrast to misonidazole and certain other fluorinated analogues, its distribution into brain tissue was poor (AUCbrain/AUCplasma = 0.07), suggesting potentially lower toxicity in spite of its higher lipophilicity (P = 0.43 versus 0.63, respectively). The bioavailability of SR-4554 from i.p. and p.o. routes was 100 and 96% respectively. In non-tumor-bearing mice, SR-4554 was excreted mainly as unchanged drug. The percentage of the injected i.p. dose of SR-4554 excreted unchanged in the urine over 24 h was 68 +/- 8%. Neither SR-4554 nor its metabolites were detected in mouse feces. We propose that these favorable pharmacokinetic properties of SR-4554 are due to the hydrophilic character and hydrogen-bonding capability of the amide and hydroxyl functions in the compound.

Animals↗
PubMed1991

Molecular epidemiology of Shigella infections: plasmid profiles, serotype correlation, and restriction endonuclease analysis.

Plasmid isolation was used to refine the epidemiologic analysis for 168 shigellosis cases in Pima County, Ariz. Plasmids of less than 20 kb were used for comparison of plasmid profiles. Plasmid patterns for each species were distinct. A total of 57 of 74 (77%) Shigella flexneri strains could be placed into seven plasmid patterns, 70 of 79 (89%) Shigella sonnei strains could be placed into seven patterns, 12 Shigella boydii strains could be placed into six patterns, and each of 3 Shigella dysenteriae strains differed. There was a correlation between plasmid patterns and serotypes for S. flexneri, and multiple plasmid patterns were found in serotypes 1, 2, and 6, offering a refinement beyond serotyping. In previous studies we found an association between Mexican travel and an S. sonnei 5.1-kb plasmid. When this plasmid was used as a probe, strong homology was seen with numerous small plasmids in all Shigella species: restriction endonuclease analysis revealed a 1.1-kb AvaI-AvaII fragment common to various plasmids of S. sonnei. S. flexneri, and S. boydii independent of species. Of 34 Pima County Shigella isolates from the mid-1970s. 8 showed plasmid patterns similar to those of the recent isolates. Some plasmids from S. sonnei, S. flexneri, and S. boydii strains isolated in the 1970s also contained the AvaI-AvaII fragment. The conservation of this specific fragment in our population for more than 12 years suggests that it may contain genes important in virulence or survival.

Arizona↗
PubMed1991

Haemophilus influenzae: an important cause of maternal and neonatal infections.

Although Haemophilus influenzae is recognized as a major pathogen of infants, its role in maternal and neonatal infections is not as well appreciated. We analyzed the records of all mothers and neonates infected with H influenzae over a 10-year period. Twenty-eight mother/neonate sets were identified in which at least one had documented infection with H influenzae. Of the 18 mothers with documented infection, 13 had chorioamnionitis, endometritis, or both, and two of these mothers were bacteremic with H influenzae. Of the 23 infected neonates, 15 presented with early sepsis and/or pneumonia and nine had conjunctivitis. During the period of the study, only group B streptococci and Escherichia coli were more common as causes of early neonatal bacteremia. Under the conditions of this retrospective study, maternal infection predicted neonatal infection. However, prospective studies in which asymptomatic patients are cultured will be required to determine how well maternal colonization/infection with H influenzae predicts neonatal infection.

Chorioamnionitis↗