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M Braddock

Publications and source records attributed to M Braddock.

At least 37 records · Page 2Linked to original sources
PubMed1995

Real-time analysis of the transcriptional regulation of HIV and hCMV promoters in single mammalian cells.

The regulation of human cytomegalovirus (hCMV) and human immunodeficiency virus (HIV) gene expression has been studied in single intact mammalian cells. Viral promoters were placed upstream of the firefly luciferase reporter gene and the resulting hybrid reporter constructs were stably integrated into the HeLa cell genome. A highly sensitive photon-counting camera system was used to study the level of gene expression in single intact cells. Luciferase expression was studied in the absence of activators of viral gene expression, in the presence of the HIV-1 TAT transactivator protein, or in the presence of sodium butyrate, a non-viral activator of gene expression. In the absence of any activator of gene expression, while expression was undetectable in most cells, significant levels of basal luciferase activity were observed in a few cells, indicating heterogeneity in gene expression in the cell population. In the presence of the general activator of viral gene expression, sodium butyrate, transcriptional activation from the viral promoters gave rise to significant and relatively homogeneous levels of luciferase expression in a majority of cells. The luciferase imaging technology was used for the real-time analysis of changes of gene expression within a single cell. This non-invasive reporter assay should become important for studies of the temporal regulation of gene expression in single cells.

Animals↗
PubMed1994

Intron-less RNA injected into the nucleus of Xenopus oocytes accesses a regulated translation control pathway.

The translation of a capped, polyadenylated RNA after injection into the nucleus of Xenopus oocytes occurs only if the RNA contains an intron. A single point mutation in the splice donor site prevents translation. Intron-less RNA is exported efficiently to the cytoplasm and is held, undegraded, in a translationally inert state for several days. Translation can be activated by treating the oocytes with progesterone or by injecting antibodies that bind the FRGY2 class of messenger RNA binding proteins, p56 and p60, but these antibodies are only effective if delivered to the nucleus. Inhibitors of casein kinase II also activate translation whereas phosphatase inhibitors block progesterone-mediated activation of translation. These data suggest the presence of an RNA handling pathway in the nucleus of Xenopus oocytes which is regulated by casein kinase type II phosphorylation and which directs transcripts to be sequestered by p56/p60 or by closely related proteins. This pathway can be bypassed if the RNA contains an intron and it can be reversed by progesterone treatment. These data may have implications for understanding translational control during early development.

Animals↗
PubMed1994

Identification of a novel HIV-1 TAR RNA bulge binding protein.

The Tat protein binds to TAR RNA to stimulate the expression of the human immunodeficiency virus type 1 (HIV-1) genome. Tat is an 86 amino acid protein that contains a short region of basic residues (aa49-aa57) that are required for RNA binding and TAR is a 59 nucleotide stem-loop with a tripyrimidine bulge in the upper stem. TAR is located at the 5' end of all viral RNAs. In vitro, Tat specifically interacts with TAR by recognising the sequence of the bulge and upper stem, with no requirement for the loop. However, in vivo the loop sequence is critical for activation, implying a requirement for accessory cellular TAR RNA binding factors. A number of TAR binding cellular factors have been identified in cell extracts and various models for the function of these factors have been suggested, including roles as coactivators and inhibitors. We have now identified a novel 38 kD cellular factor that has little general, single-stranded or double-stranded RNA binding activity, but that specifically recognises the bulge and upper stem region of TAR. The protein, referred to as BBP (bulge binding protein), is conserved in mammalian and amphibian cells and in Schizosaccharomyces pombe but is not found in Saccharomyces cerevisiae. BBP is an effective competitive inhibitor of Tat binding to TAR in vitro. Our data suggest that the bulge-stem recognition motif in TAR is used to mediate cellular factor/RNA interactions and indicates that Tat action might be inhibited by such competing reactions in vivo.

Animals↗
PubMed1994

Accuracy of fatal motorcycle-injury reporting on death certificates.

This study identifies differences in motorcycle injury fatality statistics gathered from different sources. Police Accidents Reports (PARs), identifying fatal motorcycle injuries occurring in Connecticut during 1987 were matched with state death certificates. Matched death certificates were analyzed in three major areas: content, coding, and motorcycle fatality reporting. Death certificates underreported motorcycle fatalities by 38% compared to PARs. Forty percent of death certificates were missing some or all of the required information: 7 did not include the word motorcycle, 18 did not contain acceptable ICD-9 terminology for a motorcyclist, and 17 did not describe how the injury occurred. Forty-one percent of death certificates contained external cause of injury code (E-code) errors. Incomplete information on death certificates was responsible for 52% of inaccurate reporting and E-code errors for 48%. The accuracy of fatal motorcycle injury cause of death reporting on death certificates could be improved by better physician training and rapid implementation of both the computerized death certificate coding systems and upcoming ICD-10 classification system.

Accidents, Traffic↗
PubMed1994

Burn injuries among children in an urban emergency department.

To determine the profile of burn injury among an ambulatory population, we conducted a retrospective chart review over a one-year period of all first visits by patients under age 18 receiving burn care in our emergency department. Of the 109 visits, contact burns were most numerous (43.1%) followed by scalds (33.9%), flame/explosion (11.0%), cigarettes (5.5%) and electrical (2.8%). For children under 11 years of age, contact burns caused over half of all burns. Irons accounted for 44.7% of contact burns and 19.1% of all burns. The next most frequent causative agents were beverages (14.7%), food (nonbeverage) (7.3%), tap water/bath (7.3%), and cooking-related (6.4%). Contact burns were more likely to be smaller and more localized when compared with other burns. The cause and causative agents of burns differ in frequency and relative importance depending on the patient population studied and the level of medical care being provided. Specific burn prevention strategies should be directed toward particular patterns of injury within defined patient groups.

Burns↗
PubMed1994

Inhibition of human immunodeficiency virus type 1 Tat-dependent activation of translation in Xenopus oocytes by the benzodiazepine Ro24-7429 requires trans-activation response element loop sequences.

Two benzodiazepine compounds, [7-chloro-5-(2-pyrryl)-3H-1,4 benzodiazapin-2-(H)-one] (Ro5-3335) and [7-chloro-5-(1H-pyrrol-2-yl)-3H-benzo[e] [1,4] diazepin-2-yl]- methylamine (Ro24-7429), inhibit human immunodeficiency virus type 1 (HIV-1) replication via a specific effect on the function of the transactivator protein, Tat. To gain further insight into the mechanism of action of these compounds, we have tested their effects in an alternative assay for Tat activation in Xenopus oocytes. In this system, translation of trans-activation response element (TAR)-containing RNA is activated by Tat. Both compounds specifically blocked activation of translation in a dose-dependent fashion, with Ro24-7429 showing the greater potency. In the Xenopus oocyte system, as in mammalian cells, mutation of the TAR loop sequences abolishes Tat action. However, it is possible to obtain TAR-specific, Tat-dependent activation of a target RNA with a mutation in the loop provided that this target is in large excess. This result has been interpreted as indicating that a negative factor has been titrated (M. Braddock, R. Powell, A.D. Blanchard, A.J. Kingsman, and S.M. Kingsman, FASEB J. 7:214-222, 1993). Interestingly Ro24-7429 was unable to inhibit the TAR-specific but loop sequence-independent mode of translational activation. This finding suggests that a specific loop-binding cellular factor may mediate the effects of this inhibitor of Tat action. Consistent with this notion, we could not detect any effect of Ro24-7429 on the efficiency of specific Tat binding to TAR in vitro.

Animals↗
PubMed1994

Orientation-specific cis complementation by bulge- and loop-mutated human immunodeficiency virus type 1 TAR RNAs.

Tat activates human immunodeficiency type 1 gene expression by binding to TAR RNA. TAR comprises a partially base paired stem and hexanucleotide loop with a tripyrimidine bulge in the upper stem. In vitro, Tat binds to the bulge and upper stem, with no requirement for the loop. However, in vivo, loop sequences are critical for activation, implying that a loop binding cellular factor may be involved in the activation pathway. Given that activation appears to be a two-component system comprising a Tat-bulge interaction and a cellular factor-loop interaction, we considered that it might be possible to spatially separate the two components and retain activation. We have constructed a series of double TAR elements comprising various combinations of mutated TAR structures. Defective TARs with nucleotide substitutions in either the bulge or the loop complemented each other to give wild-type activation. However, the complementation was orientation specific, requiring the intact Tat binding site to reside on the 5'-proximal TAR. These data suggest that provided the wild-type orientation of the bulge and loop elements is retained, there is no requirement for them to coexist on the same TAR structure.

Bacterial Proteins↗
PubMed1994

Using a geographic information system to understand child pedestrian injury.

Data from police accident reports involving pedestrians less than 20 years of age in Hartford, Conn, during 1988 through 1990 were abstracted and entered into a geographic information system. Two high-frequency collision areas were identified and compared. There were 374 child pedestrians involved in collisions (a rate of 28 per 10,000). Two high-occurrence areas accounted for 30% of collisions. Collisions in one of these areas were more likely to involve younger children (8.1 vs 10.2 years of age) and to occur in the late afternoon, and occurred closer to the child's residence, than collisions in the other area. The geographic information system is a useful tool in the study of child pedestrian collisions.

Accidents, Traffic↗
PubMed1993

Enhanced activity of human G6PD promoter transfected in HeLa cells producing high levels of HIV-1 Tat.

Glucose 6-phosphate dehydrogenase (G6PD) enzyme activity has an important role in the cell defence against oxidative damage produced by hydrogen peroxide, a compound known to activate HIV-1 expression through the intermediate of NF-kappa B. By means of CAT assays, in this paper we show that the Tat protein increases the rate of transcription from the human G6PD promoter in HeLa cells; furthermore, we report a similar effect of Tat on transcription driven from the viral RSV promoter. We did not observe Tat stimulation with the human CMV, SV40 or the basic RSV promoters. Dose-response curves indicate that Tat activates G6PD and RSV through a mechanism different from the major one operating on the HIV-1 LTR promoter. TAR-like structures are not involved, instead a short sequence close to the G6PD transcription start site and the RSV LTR enhancer is a good candidate for mediating the phenomena described in this paper. This sequence has some features in common with the NF-kappa B motif.

Base Sequence↗
PubMed1993

HIV-1 TAR RNA-binding proteins control TAT activation of translation in Xenopus oocytes.

Human immunodeficiency virus (HIV-1) gene expression is activated by the viral TAT protein that interacts with an RNA sequence, TAR, located at the 5' end of all viral mRNAs. TAT functions primarily as a transcriptional activator in mammalian cells. However, in Xenopus oocytes TAT functions primarily as a translational activator. TAR is an RNA structure comprising a partially base-paired stem, a tripyrimidine bulge in the upper stem, and an unpaired six-nucleotide loop. In vitro, TAT binds directly to the bulge with no requirement for the loop. In vivo, however, mutations in the loop abolish TAT activation of transcription and translation, implying a requirement for TAR-binding cellular factors. We now provide genetic evidence for the presence of two TAR-specific cellular factors in Xenopus oocytes. These factors display independent and mutually exclusive interactions with either the loop or the bulge region of TAR. Furthermore, by using in vivo RNA competition assays we show that the cellular factors regulate the accessibility of the TAT binding site. The fact that Xenopus oocytes contain factors that specifically interact with a human viral RNA sequence might indicate that the TAT/TAR interaction is subverting a conserved pathway in the cell.

Animals↗
PubMed1992

A population-based study of motorcycle injury and costs.

STUDY OBJECTIVE: To provide a population-based injury and cost profile for motorcycle injury in Connecticut. DESIGN: Population-based retrospective epidemiologic review of Connecticut death certificates, hospital discharge data, and police accident reports. RESULTS: Connecticut death certificates identified 112 deaths from motorcycle injuries for an annual death rate of 1.2 per 100,000 persons. Death rates were highest among 20- to 24-year-old men. Nonhelmeted motorcyclists were 3.4-fold more likely to die than were helmeted riders (P less than .05). An estimated 2,361 motorcycle-related hospital discharges resulted in an annual hospitalization rate of 24.7 per 100,000 persons. Head, neck, and spinal injuries accounted for 22% of all injuries. Total costs exceeded $29 million; 29% of hospitalized patients were uninsured, and 42% of the cost was not reimbursed to the hospitals. CONCLUSION: Motorcycle injuries contribute significantly to Connecticut's mortality, morbidity, and medical costs. Our study suggests that a uniform helmet law would save an estimated ten lives and prevent more than 90 nonfatal injuries in Connecticut each year at a cost savings to the state of $5.1 million. These data are crucial in advocating re-enactment of motorcycle helmet laws.

Abbreviated Injury Scale↗
PubMed1992

An adenosine at position 27 in the human immunodeficiency virus type 1 trans-activation response element is not critical for transcriptional or translational activation by Tat.

Tat protein binds to the trans-activation response (TAR) element of human immunodeficiency virus type 1 RNAs and activates gene expression at the level of transcription in mammalian cell lines and translation in Xenopus oocytes. Certain residues within TAR are important for Tat binding in vitro, including residue A-27, which appears to be able to be modified in a Tat-dependent manner in Xenopus oocytes (L. Sharmeen, B. Bass, N. Sonenberg, H. Weintraub, and M. Groudine, Proc. Natl. Acad. Sci. USA 88:8096-8100, 1991). Activation by Tat in oocytes occurs via a covalent modification of TAR-containing RNA. We have found that in both mammalian cells and Xenopus oocytes, conversion of A-27.U-38 or C-27.G-38 or C-27.G-38 reduces activation. However, conversion to G-27.U-38 or G-27.C-38 had little or no effect on activation, and in oocytes, these mutant RNAs were still covalently modified. These data exclude a specific role for the adenosine at residue 27 for Tat activation but suggest a requirement for a purine at this position.

Adenosine↗
PubMed1992

Work-related injury among Connecticut minors.

Work-related injuries have recently been shown to be a significant cause of morbidity among adolescents. This study represents a population-based work-related injury profile for Connecticut minors. Review of 796 worker compensation reports for adolescents from 14 through 17 years of age submitted over 12 months revealed an overall age-specific injury rate of 15 per 1000 employed 16- and 17-year-olds; frequency of injury increased with age. Social and recreational workers had an injury rate of more than 20%, predominantly sprains and contusions. Among all other occupations and industries, cuts were the major type of injury (34%); more than one third of cutting injuries were associated with use of case cutters, another third with knives. There were no work-related deaths among minors in this study. Minors in the workplace are at high risk of injury compared with adults. This study suggests that identification of specific patterns of work-related injury could lead to targeted intervention strategies.

Accidents, Occupational↗
PubMed1991

Blocking of Tat-dependent HIV-1 RNA modification by an inhibitor of RNA polymerase II processivity.

Human immunodeficiency virus gene expression is regulated transcriptionally and post-transcriptionally by the virally encoded tat protein (Tat). Tat functions through an RNA target sequence located in the untranslated region at the 5' end of viral transcripts. In Xenopus oocytes, translation of RNA containing the target sequence is specifically activated by Tat. This activation only occurs if the RNA is injected into the nucleus, and might be due to Tat-dependent, nucleus-specific chemical modification of the RNA which somehow facilitates translation. Here we demonstrate that Tat activation of its target RNA in the nucleus involves a Tat-dependent covalent modification. The modified RNA is competent for translation after reinjection into either the nucleus or the cytoplasm in the absence of Tat. Furthermore, we find that the nucleoside analogue 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, which inhibits processivity of RNA polymerase II (ref. 9), blocks this Tat-dependent modification.

Animals↗
PubMed1991

Child pedestrian injury: a population-based collision and injury severity profile.

Linked multiple data sources were analyzed to provide a population-based collision and injury severity profile among pedestrians under 20 years of age struck by a motor vehicle during 1986-1987 in Hartford, Connecticut. Data sources included police accident reports, medical examiner records, and hospital charts. There were 234 motor vehicle-pedestrian collisions reported to the police in the study period. Of these, 213 were Hartford residents resulting in an annual age-specific pedestrian collision rate of 22.8 per 10,000 persons. A spot map of collision location reveals several well-defined geographic areas, which includes nearly half (45%) of the motor vehicle-pedestrian collisions. We reviewed 143 of 192 medical charts (75%) and 6 medical examiner records. The case fatality rate was 4.2% and the mean Injury Severity Score was 4.4. These findings will be useful for designing, implementing, and evaluating a targeted child pedestrian safety program.

Accidents, Traffic↗
PubMed1991

Population, income, and ecological correlates of child pedestrian injury.

Pedestrian injury is a significant health problem among urban children. This study is an analysis of the role of population, income, and ecological factors in the occurrence of child pedestrian collisions. One hundred and ninety-eight motor vehicle collisions occurring in Hartford, Connecticut involving pedestrians younger than 15 years old were reported to police during 1986 through 1987. Collision locations were abstracted from police reports and assigned a census tract. Census tracts were classified as "high frequency" (8+ collisions), "moderate frequency" (3 to 7 collisions), or "low frequency" (0 to 2 collisions). High-frequency census tracts had greater proportions of children and of nonwhite residents than moderate- or low-frequency tracts. They also were characterized by high proportions of households headed by females living below the poverty line. High-frequency tracts had a greater number of children per acre than moderate or low tracts. Children per acre had the strongest association with collision frequency (R = .72) and remained the most consistent when other variables were controlled. The number of children per acre is a potentially useful predictor of census tracts at risk for child pedestrian collisions. This may be useful in developing focused prevention strategies within an urban environment.

Accidents, Traffic↗
PubMed1990

A nuclear translational block imposed by the HIV-1 U3 region is relieved by the Tat-TAR interaction.

Replication of HIV-1 depends on the viral Tat protein, which functions via a target sequence, TAR, present in the proviral long terminal repeat (LTR) and at the 5' end of viral mRNAs. We have shown that Tat potentiates the expression of TAR-containing RNAs, but only when Tat and the TAR-containing RNA are present in the nucleus. We now show that a small change in the TAR loop abolishes nuclear potentiation by Tat. Furthermore, the HIV-1 U3 region induces expression incompetence in mRNA synthesized by this promoter. RNAs of identical structure are, however, translated efficiently when produced from the CMV-IE promoter. The Tat-TAR system appears, therefore, to rescue the expression potential of HIV-1 LTR-directed RNA.

Animals↗