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Biomedical subjects

R Kelly

Publications and source records attributed to R Kelly.

At least 37 records · Page 2Linked to original sources
PubMed1998

High efficiency myogenic conversion of human fibroblasts by adenoviral vector-mediated MyoD gene transfer. An alternative strategy for ex vivo gene therapy of primary myopathies.

Ex vivo gene therapy of primary myopathies, based on autologous transplantation of genetically modified myogenic cells, is seriously limited by the number of primary myogenic cells that can be isolated, expanded, transduced, and reimplanted into the patient's muscles. We explored the possibility of using the MyoD gene to induce myogenic conversion of nonmuscle, primary cells in a quantitatively relevant fashion. Primary human and murine fibroblasts from skin, muscle, or bone marrow were infected by an E1-deleted adenoviral vector carrying a retroviral long terminal repeat-promoted MyoD cDNA. Expression of MyoD caused irreversible withdrawal from the cell cycle and myogenic differentiation in the majority (from 60 to 90%) of cultured fibroblasts, as defined by activation of muscle-specific genes, fusion into contractile myotubes, and appearance of ultrastructurally normal sarcomagenesis in culture. 24 h after adenoviral exposure, MyoD-converted cultures were injected into regenerating muscle of immunodeficient (severe combined immunodeficiency/beige) mice, where they gave rise to beta-galactosidase positive, centrally nucleated fibers expressing human myosin heavy chains. Fibers originating from converted fibroblasts were indistinguishable from those obtained by injection of control cultures of lacZ-transduced satellite cells. MyoD-converted murine fibroblasts participated to muscle regeneration also in immunocompetent, syngeneic mice. Although antibodies from these mice bound to adenoviral infected cells in vitro, no inflammatory infiltrate was present in the graft site throughout the 3-wk study period. These data support the feasibility of an alternative approach to gene therapy of primary myopathies, based on implantation of large numbers of genetically modified primary fibroblasts massively converted to myogenesis by adenoviral delivery of MyoD ex vivo.

Adenoviridae↗
PubMed1998

An improved protocol for the preparation of yeast cells for transformation by electroporation.

Pretreatment of yeast cells with lithium acetate (LiAc) and dithiothreitol (DTT) enhances the frequency of transformation by electroporation. The method shows improvements of 6-67-fold in wild-type strains derived from commonly used Saccharomyces cerevisiae genetic backgrounds. In addition, 15-300-fold improvement in transformation frequency was achieved with several mutant strains of S. cerevisiae that transformed poorly by conventional procedures. Both DTT and lithium acetate were necessary for maximal transformation frequencies. Pretreatment with lithium and DTT also resulted in an approximately 3-5-fold increase in the electroporation transformation frequency of the pathogenic fungus Candida albicans.

Blotting, Southern↗
PubMed1998

Alteration in myosatellite cell commitment with muscle maturation.

Myosatellite cells are myoblasts found between the basal lamina and sarcolemma of myofibers of postnatal mice. The extent to which these cells are programmed, upon differentiation, to express isoforms of contractile protein genes specific to the type of fiber with which they are associated has been evaluated in vitro using myosatellite cells derived from the soleus and the extensor digitorum longus muscles (EDL) of 4-day-old and adult transgenic mice, which express nuclear localizing beta-galactosidase (nlsbeta-gal) under the control of the promoter and 3' enhancer of the gene encoding fast myosin light chain 3F (MLC3F) (Kelly et al. [1995] J. Cell Biol. 129:383-396). Cultures were allowed to differentiate either as myocytes (mononucleated cells), to prevent possible modification of the myosatellite phenotype by other myonuclei in mosaic myotubes, or as myotubes. Transgene expression was age related, with 90% and 70% of the myocytes derived from the neonatal EDL and soleus muscles (muscles that had not yet achieved their mature phenotype), respectively, having nuclei encoding beta-gal; 61% and 32% of the myocyte nuclei derived from myosatellite cells of the adult EDL (a fast muscle) and the adult soleus muscle (a mixed muscle containing many slow myofibers), respectively, expressed this transgene. Because myosatellite cells found in adult muscles are the progeny of those found in the neonate, an alteration of myosatellite cell commitment to express this transgene occurs with muscle maturation. Because expression of the transgene in neonatal and adult muscle in vivo reflects the expression of the endogenous MLC3F gene (Kelly et al. [1995] J. Cell Biol. 129:383-396), it is likely that expression of the transgene by differentiated myosatellite cells reflects the extent of commitment of these cells to produce MLC3F. A hypothesis is presented that MLC3F is widely expressed in developing muscles but eliminated in myofibers that undergo maturation toward a slower phenotype.

Animals↗
PubMed1998

On human cloning.

Explore the source record for details and available documents.

Cloning, Organism↗
PubMed1998

How valid are medical records and patient questionnaires for physician profiling and health services research? A comparison with direct observation of patients visits.

OBJECTIVES: This study was designed to determine the optimal nonobservational method of measuring the delivery of outpatient medical services. METHODS: As part of a multimethod study of the content of primary care practice, research nurses directly observed consecutive patient visits to 138 practicing family physicians. Data on services delivered were collected using a direct observation checklist, medical record review, and patient exit questionnaires. For each medical service, the sensitivity, specificity, and Kappa statistic were calculated for medical record review and patient exit questionnaires compared with direct observation. Interrater reliability among eight research nurses was calculated using the Kappa statistic for a separate sample of videotaped visits and medical records. RESULTS: Visits by 4,454 patients were observed. Exit questionnaires were returned by 74% of patients. Research nurse interrater reliabilities were generally high. The specificity of both the medical record and the patient exit questionnaire was high for most services. The sensitivity of the medical record was low for measuring health habit counseling and moderate for physical examination, laboratory testing, and immunization. The patient exit questionnaire showed moderate to high sensitivity for health habit counseling and immunization and variable sensitivity for physical examination and laboratory services. CONCLUSIONS: The validity of the medical record and patient questionnaire for measuring delivery of different health services varied with the service. This report can be used to choose the optimal nonobservational method of measuring the delivery of specific ambulatory medical services for research and physician profiling and to interpret existing health services research studies using these common measures.

Adult↗
PubMed1998

Differential activation of Myf5 and MyoD by different Wnts in explants of mouse paraxial mesoderm and the later activation of myogenesis in the absence of Myf5.

Activation of myogenesis in newly formed somites is dependent upon signals derived from neighboring tissues, namely axial structures (neural tube and notochord) and dorsal ectoderm. In explants of paraxial mesoderm from mouse embryos, axial structures preferentially activate myogenesis through a Myf5-dependent pathway and dorsal ectoderm preferentially through a MyoD-dependent pathway. Here we report that cells expressing Wnt1 will preferentially activate Myf5 while cells expressing Wnt7a will preferentially activate MyoD. Wnt1 is expressed in the dorsal neural tube and Wnt7a in dorsal ectoderm in the early embryo, therefore both can potentially act in vivo to activate Myf5 and MyoD, respectively. Wnt4, Wnt5a and Wnt6 exert an intermediate effect activating both Myf5 and MyoD equivalently in paraxial mesoderm. Sonic Hedgehog synergises with both Wnt1 and Wnt7a in explants from E8.5 paraxial mesoderm but not in explants from E9.5 embryos. Signaling through different myogenic pathways may explain the rescue of muscle formation in Myf5 null embryos, which do not form an early myotome but later develop both epaxial and hypaxial musculature. Explants of unsegmented paraxial mesoderm contain myogenic precursors capable of expressing MyoD in response to signaling from a neural tube isolated from E10.5 embryos, the developmental stage when MyoD is present throughout the embryo. Myogenic cells cannot activate MyoD in response to signaling from a less mature neural tube. Together these data suggest that different Wnt molecules can activate myogenesis through different pathways such that commitment of myogenic precursors is precisely regulated in space and time to achieve the correct pattern of skeletal muscle development.

Animals↗
PubMed1998

An ecological study of the effectiveness of mammography in reducing breast cancer mortality.

OBJECTIVES: The purpose of this study was to determine the relation of screening mammography to breast cancer incidence and case fatality. METHODS: In a sample of White female Medicare beneficiaries hospitalized in 1990-1991, age-adjusted breast cancer incidence and 2-year case fatality rates were estimated and compared with the frequency of mammographic screening from a population-based survey. RESULTS: The average rates for incidence, case fatality, and mammography within 5 years in 29 states were 414/100,000, 18.8%, and 59.2%, respectively. There was a positive state-level correlation between mammography rates and incidence and an inverse correlation between mammography and case fatality. CONCLUSIONS: High screening mammography rates in some states are associated with reduced breast cancer case fatality rates, presumably as a result of diagnosis of earlier stage cancers.

Aged↗
PubMed1998

The influence of amniotic fluid on prostaglandin synthesis and metabolism in human fetal membranes.

OBJECTIVE: To investigate the effect of amniotic fluid on prostaglandin synthesis and metabolism in the fetal membranes. DESIGN: A cell culture study of amnion and chorion obtained at elective cesarean section incubated with amniotic fluid collected following either spontaneous labor and delivery, or elective cesarean section. SUBJECTS: Forty-eight pregnant women at 3742 weeks gestation: 24 in spontaneous labor and 24 delivered by elective cesarean section. RESULTS: Significantly more PGE2 and PGF2alpha were produced by amnion and chorion treated with amniotic fluid from spontaneous labor compared with elective cesarean section. Spontaneous labor amniotic fluid favors PGE2 and PGFM production by amnion and chorion respectively; while elective section fluid stimulates PGE2 synthesis by both tissues (reflected as PGEM in chorion). Amniotic fluid, from either spontaneous labor or elective section, had no effect on the metabolism of exogenous PGE2 or PGF2alpha by chorion cells. CONCLUSION: Spontaneous labor is associated with the presence of a substance in amniotic fluid which facilitates prostaglandin synthesis in the fetal membranes, but which is without effect on prostaglandin metabolism.

Amnion↗
PubMed1997

Regionalized transcriptional domains of myosin light chain 3f transgenes in the embryonic mouse heart: morphogenetic implications.

Within the embryonic heart, five segments can be distinguished: two fast-conducting atrial and ventricular compartments flanked by slow-conducting segments, the inflow tract, the atrioventricular canal, and the outflow tract. These compartments assume morphological identity as a result of looping of the linear heart tube. Subsequently, the formation of interatrial, interventricular, and outflow tract septa generates a four-chambered heart. The lack of markers that distinguish right and left compartments within the heart has prevented a precise understanding of these processes. Transgenic mice carrying an nlacZ reporter gene under transcriptional control of regulatory sequences from the MLC1F/3F gene provide specific markers to investigate such regionalization. Our results show that transgene expression is restricted to distinct regions of the myocardium: beta-galactosidase activity in 3F-nlacZ-2E mice is confined predominantly to the embryonic right atrium, atrioventricular canal, and left ventricle, whereas, in 3F-nlacZ-9 mice, the transgene is expressed in both atrial and ventricular segments (right/left) and in the atrioventricular canal, but not in the inflow and outflow tracts. These lines of mice illustrate that distinct embryonic cardiac regions have different transcriptional specificities and provide early markers of myocardial subdivisions. Regional differences in transgene expression are not detected in the linear heart tube but become apparent as the heart begins to loop. Subsequent regionalization of transgene expression provides new insights into later morphogenetic events, including the development of the atrioventricular canal and the fate of the outflow tract.

Animals↗
PubMed1997

Emergence of further serotypes of multiple drug-resistant Streptococcus pneumoniae in Queensland.

We describe 27 cases of multiple drug-resistant pneumococcal infection in Queensland children (7 cases) and adults (20 cases), between February 1995 and October 1996. Seven patients had invasive disease. Serotypes were those commonly associated with paediatric infections and included types 19F (15 strains), 14 (6), 23F (4), 6A (1) and 19A (1). No rifampicin or vancomycin resistance was encountered. However, pneumococci fully resistant to cotrimoxazole, erythromycin and tetracycline were isolated from 25 of 27 cases (93%). Strains with high level resistance to penicillin and chloramphenicol were also recovered from 16 (59%) and 19 (70%) patients respectively. Twelve of 16 penicillin-resistant isolates showed intermediate resistance to ceftriaxone and two strains were fully resistant to this antibiotic. Clones of types 19F and 14 pneumococci, each with two distinctive resistance patterns, appear to be established in south-east Queensland.

Adult↗
PubMed1997

Persistence in muscle of an adenoviral vector that lacks all viral genes.

Genetic correction of inherited muscle diseases, such as Duchenne muscular dystrophy, will require long term expression of the recombinant protein following gene transfer. We have shown previously that a new adenoviral vector that lacks all viral genes expressed both full-length dystrophin and beta-galactosidase in mdx (dystrophin-deficient) mouse muscle. We observed a significant histologic improvement of vector-transduced mdx muscle before the eventual loss of vector-encoded transgene expression. In this study, we investigated whether an immunological response against vector-encoded beta-galactosidase contributed to the loss of vector expression and affected vector persistence in muscle. Intramuscular vector injection in control normal mice resulted in an early and complete loss of beta-galactosidase expression accompanied by predominantly CD4+ and CD8+ lymphocytic infiltration and a significant loss of vector DNA. In contrast, intramuscular vector injection in lacZ transgenic mice resulted in persistent expression of beta-galactosidase for at least 84 days with no evidence of inflammation or significant loss of vector DNA. Our studies demonstrate that, in the absence of an immune response induced by beta-galactosidase expression, an adenoviral vector lacking all viral genes is stably maintained in muscle.

Adenoviridae↗
PubMed1997

Limitations of nls beta-galactosidase as a marker for studying myogenic lineage or the efficacy of myoblast transfer.

BACKGROUND: Nuclear localizing beta-galactosidase (nls beta-gal) is used as a marker for studying myoblast cell lineage and for evaluating myoblast survival after myoblast transfer, a procedure with potential use for gene complementation for muscular dystrophy. Usefulness of this construct depends on the establishment of the extent to which nls beta-gal or its mRNA may be translocated from the nucleus that encodes it to other non-coding myonuclei in hybrid myofibers and the ease with which the encoding and non-coding myonuclei can be distinguished. Previous in vitro studies (Ralston and Hall 1989. Science, 244:1066-1068) have suggested limited translocation of the fusion protein. We re-examined the extent to which nls beta-gal is translocated in hybrid myofibers, both in vitro and in vivo, and evaluated the extent to which one can rely on histochemistry to distinguish encoding from non-coding nuclei in these myofibers. METHODS: Myotubes formed in co-cultures of a myoblast line (MM14 cells), stably transfected with a construct consisting of a nls beta-gal under the control of the myosin light chain 3F promoter and 3' enhancer (3FlacZ10 cells), and [3H]-thymidine-labeled parental MM14 cells (plated at ratios of 1:6 or 1:20, respectively) were reacted with X-gal. After autoradiography, the distance over which nls beta-gal was translocated in hybrid myotubes was determined. In vivo translocation of nls beta-gal was evaluated by injecting [3H]-thymidine-labeled 3FlacZ10 myoblasts into the regenerating extensor digitorum longus muscle of immunosuppressed normal and mdx (dystrophin deficient) mice. Sections stained with X-gal and subjected to autoradiography permitted determination of the extent of nls beta-gal translocation in hybrid myofibers. RESULTS: In vitro: All nuclei in > 92% of hybrid myotubes showed evidence of nls beta-gal after exposure to X-gal, suggesting extensive translocation. Within hybrid myotubes, MM14-derived myonuclei approximately 350 microns from a 3FlacZ10-derived myonucleus showed evidence of nls beta-gal. In vivo: Similar translocation of nls beta-gal was observed in vivo. One week after myoblast transfer, donor-derived myonuclei were distinguishable from host-derived myonuclei containing nls beta-gal by the greater accumulation of reaction product in donor myonuclei after X-gal staining. However, 2 weeks after injection, host myonuclei often contained a significant amount of nls beta-gal, and accumulation of reaction product could not be used as the criterion for identification of donor myonuclei. CONCLUSIONS: Translocation of nls beta-gal (or its mRNA) is significantly greater than previously reported (Ralston and Hall 1989), resulting in large numbers of nls beta-gal positive non-coding myonuclei in hybrid myofibers. One week after myoblast transfer, distinguishing between nls beta-gal encoding and non-coding myonuclei in hybrid myofibers after X-gal staining of sectioned muscle is feasible; however, by 2 weeks, nls beta-gal increases in host myonuclei, making identification of donor-derived myonuclei problematic. Translocation of nls beta-gal to non-coding myonuclei in hybrid myofibers must be considered when nls beta-gal is used for studies of myogenic lineage or the efficacy of myoblast transfer therapy, particularly if long-term survival of hybrid myotubes is required.

Animals↗
PubMed1997

Fibre type-specific and nerve-dependent regulation of myosin light chain 1 slow promoter in regenerating muscle.

The regulation of a slow muscle gene, the myosin light chain 1 slow/ventricular gene, has been studied by in vivo transfection into regenerating rat skeletal muscle. Constructs containing portions of the myosin light chain 1 slow/ventricular promoter linked to reporter genes were injected into fast and slow muscles 3 days after muscle injury by bupivacaine injection, and reporter gene activity was analysed after 10 days. We report that a sequence in the 5' flanking region of the myosin light chain 1 slow/ventricular gene is able to direct slow muscle-specific regulation of reporter genes, and that the expression of the transgene, like that of the corresponding endogenous gene, is dependent on intact nerve. This study validates the use of regenerating muscle as a model for studying muscle gene regulation and is the first demonstration of a myosin gene promoter regulated by nerve activity.

Animals↗
PubMed1997

Adolescents, sexual behaviour and HIV-1 in rural Rakai district, Uganda.

OBJECTIVES: To describe the epidemiology of HIV-1 infection among adolescents aged 13-19 years, in rural Rakai district, Uganda. STUDY DESIGN: Baseline survey and 2-year follow-up (1990-1992) of adolescents in a population-based, open rural cohort. METHODS: Annual enumeration and behavioral/serological survey of all consenting adolescents aged 13-19 years at recruitment, residing in 31 randomly selected community clusters. RESULTS: At baseline, of 909 adolescents present in study clusters, 824 (90.6%) provided interview data and serological samples. No adolescents aged 13-14 years were HIV-infected. Among those aged 15-19 years, 1.8% of men and 19.0% of women were HIV-positive. Among young women aged 15-19 years in marital/consensual union, 21.3% were HIV-positive; this rate did not differ significantly from the 29.1% prevalence in those reporting non-permanent relationships; prevalence was significantly lower in women reporting no current relationship (4.3%). After multivariate adjustment, female sex, age 17-19 years, residence in trading centers/trading villages and a history of sexually transmitted disease symptoms remained significantly associated with HIV infection. Seventy-nine per cent of adolescents provided a follow-up serological sample. No young men aged 13-14 years seroconverted during the study; in young women aged 13-14 years, HIV seroincidence was 0.6 per 100 person-years (PY) of observation. Among young men aged 15-19 years, there were 1.1 +/- 0.6 seroconversions per 100 PY of observation prior to age 21 years; among women 15-19 years, the incidence rate was 3.9 +/- 1.0 per 100 PY of observation prior to age 21 years. The mortality rate among HIV-positive adolescents aged 15-19 years, at 3.9 per 100 PY of observation, was 13-fold higher than that among the HIV-uninfected. By 1992, knowledge of sexual transmission was almost universal, the proportions reporting multiple partners had decreased and condom use had increased over baseline. CONCLUSIONS: Adolescents, and young women in particular, are vulnerable to HIV infection. Despite reported behavioral changes, HIV incidence rates remain substantial, and there is a need for innovative HIV preventive measures.

Adolescent↗