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Biomedical subjects

J Turner

Publications and source records attributed to J Turner.

At least 163 records · Page 9Linked to original sources
PubMed1996

Dynamics of loading the beta sliding clamp of DNA polymerase III onto DNA.

A "minimal" DNA primer-template system, consisting of an 80-mer template and 30-mer primer, supports processive DNA synthesis by DNA polymerase III core in the presence of the beta sliding clamp, gamma complex clamp loader, and single-stranded binding protein from Escherichia coli. This primer-template system was used to measure the loading of the beta sliding clamp by the gamma complex in an ATP-dependent reaction. Bound protein-DNA complexes were detected by monitoring fluorescence depolarization of DNA. Steady state and time-resolved anisotropies were measured, and stopped-flow pre-steady state fluorescence measurements allowed visualization of the loading reactions in real time. The rate of loading beta onto DNA was 12 s-1, demonstrating that clamp assembly is rapid on the time scale required for lagging strand Okazaki fragment synthesis. The association rate appears to be limited by an intramolecular step occurring prior to the clamp-loading reaction, possibly the opening of the toroidal beta dimer.

Adenosine Triphosphate↗
PubMed1996

Replisome assembly reveals the basis for asymmetric function in leading and lagging strand replication.

The E. coli replicase, DNA polymerase III holoenzyme, contains two polymerases for replication of duplex DNA. The DNA strands are antiparallel requiring different modes of replicating the two strands: one is continuous (leading) while the other is discontinuous (lagging). The two polymerases within holoenzyme are generally thought to have asymmetric functions for replication of these two strands. This report finds that the two polymerases have equal properties, both are capable of replicating the more difficult lagging strand. Asymmetric action is, however, imposed by the helicase that encircles the lagging strand. The helicase contact defines the leading polymerase constraining it to a subset of actions, while leaving the other to cycle on the lagging strand. The symmetric actions of the two polymerases free holoenzyme to assemble into the replisome in either orientation without concern for a correct match to one or the other strand.

Bacterial Proteins↗
PubMed1996

Particle-mediated gene transfer of granulocyte-macrophage colony-stimulating factor cDNA to tumor cells: implications for a clinically relevant tumor vaccine.

The necessity for prolonged tissue culture manipulations limits the clinical application of many form of gene therapy in patients with malignancies. We hypothesized that granulocyte-macrophage colony-stimulating factor (GM-CSF) cDNA in a plasmid expression vector could be effectively introduced into resting tumor cells, without the need for tissue culture propagation prior to or following transfection, and that efficient expression of transgenic GM-CSF by the transfected tumor cells would confer an effective immune response against tumors. GM-CSF cDNA in expression vectors was coated onto gold particles and accelerated with a gene gun device into mouse and human tumor cells. Human tumor tissue transfected within 4 hr of surgery produced significant levels of transgenic human GM-CSF protein in vitro. Human GM-CSF was readily detectable in serum and at the injection site following subcutaneous implantation of these transfected tumor cells into nude mice. Transfected and irradiated murine B16 melanoma cells produced > or = 100 ng/ml murine GM-CSF/10(6) cells per 24 hr in vitro for at least 10 days. The antitumor efficacy of this nonviral approach was tested using irradiated B16 tumor cells that were transfected with mGM-CSF cDNA and injected into mice as tumor "vaccine". Subsequent challenge of these mice with nonirradiated, nontransfected B16 tumor cells showed that 58% of the animals wer protected from the tumor by the prior vaccine treatment. In contrast, only 2% of control animals were protected by prior treatment with irradiated B16 cells transfected with the vector containing the luciferase gene. These results suggest that particle-mediated transfection of fresh tumor explants with cytokine cDNA is an effective and clinically attractive approach for cancer therapy.

Animals↗
PubMed1996

Efficacy of a small single dose of oral dexamethasone for outpatient croup: a double blind placebo controlled clinical trial.

OBJECTIVE: To assess the efficacy of a single dose of oral dexamethasone 0.15 mg/kg in children with mild croup not admitted to hospital. DESIGN: Double blind, randomised, placebo controlled clinical trial. SETTING: The emergency department of a tertiary paediatric hospital. SUBJECTS: 100 children aged 4-122 months presenting with mild croup. INTERVENTION: A single oral dose of dexamethasone 0.15 mg/kg or placebo. MAIN OUTCOME MEASURE: Return to medical care with ongoing croup. RESULTS: Baseline characteristics of the two treatment groups were similar. Eight children (all from the placebo group) returned to medical care with ongoing croup, one being admitted. There was no reported difference in duration of croup symptoms, duration of viral symptoms, or rate of return to medical care for other reasons. CONCLUSION: Oral dexamethasone in a dose of 0.15 mg/kg is effective in reducing return to medical care with ongoing croup in children with mild croup.

Administration, Oral↗
PubMed1996

Gene gun-mediated skin transfection with interleukin 12 gene results in regression of established primary and metastatic murine tumors.

Particle-mediated (gene gun) in vivo delivery of the murine interleukin 12 (IL-12) gene in an expression plasmid was evaluated for antitumor activity. Transfer of IL-12 cDNA into epidermal cells overlying an implanted intradermal tumor resulted in detectable levels (266.0 +/- 27.8 pg) of the transgenic protein at the skin tissue treatment site. Despite these low levels of transgenic IL-12, complete regression of established tumors (0.4-0.8 cm in diameter) was achieved in mice bearing Renca, MethA, SA-1, or L5178Y syngeneic tumors. Only one to four treatments with IL-12 cDNA-coated particles, starting on day 7 after tumor cell implantation, were required to achieve complete tumor regression. This antitumor effect was CD8+ T cell-dependent and led to the generation of tumor-specific immunological memory. By using a metastatic P815 tumor model, we further showed that a delivery of IL-12 cDNA into the skin overlying an advanced intradermal tumor, followed by tumor excision and three additional IL-12 gene transfections, could significantly inhibit systemic metastases, resulting in extended survival of test mice. These results suggest that gene gun-mediated in vivo delivery of IL-12 cDNA should be further developed for potential clinical testing as an approach for human cancer gene therapy.

Animals↗
PubMed1996

Beta-amyloidosis in normal aging and transmitter signaling in human temporal lobe.

Interactions between abnormal amyloid precursor protein metabolism and cholinergic dysfunction are increasingly apparent. Both of these major features of Alzheimer's disease occur in restricted loci in normal aging--a potential model for early Alzheimer type pathology. Entorhinal cortex is particularly vulnerable to beta-amyloidosis and compared with other cortical areas is remarkable for the relatively high density of nicotinic (3H-nicotine) but not other cholinergic or glutamate receptor binding. With increasing age, post-maturity, there is a persistent decline in nicotinic receptor binding in entorhinal cortex whereas muscarinic M1 and non-M1, glutamate NMDA and non-NMDA receptors are spared. Normal elderly individuals, distinguished by the absence of beta A4 immunoreactive plaques in this area, are differentiated from those with plaques by higher nicotine binding. Amongst individuals with an established history of smoking tobacco, nicotinic receptor binding and hippocampal choline acetyltransferase were elevated compared with non-smokers and preliminary evidence indicates a reduced density of cortical plaques. These findings are consistent with the hypothesis that down regulation of the nicotinic cholinergic receptor--a ligand gated calcium channel known to control the expression of neurotrophins--plays a role in the evolution of Alzheimer-type pathology.

Adolescent↗
PubMed1996

A molecular switch in a replication machine defined by an internal competition for protein rings.

Replication machines use ring-shaped clamps that encircle DNA to tether the polymerase to the chromosome. The clamp is assembled on DNA by a clamp loader. This report shows that the polymerase and clamp loader coordinate their actions with the clamp by competing for it through overlapping binding sites. The competition is modulated by DNA. In the absence of DNA, the clamp associates with the clamp loader. But after the clamp is placed on DNA, the polymerase develops a tight grip on the clamp and out-competes the clamp loader. After replication of the template, the polymerase looses affinity for the clamp. Now the clamp loader regains access to the clamp and removes it from DNA thus recycling it for future use.

Binding, Competitive↗
PubMed1996

Viral load, CD4 percentage, and delayed-type hypersensitivity in subjects receiving the HIV-1 immunogen and antiviral drug therapy.

Two trials of subjects inoculated with the inactivated, gp120-depleted HIV-1 Immunogen are reported. In one study, in which 19 subjects received ZDV and 8 subjects received ddI, treatment with the HIV-1 Immunogen did not affect the pharmacokinetic parameters of the antiviral drugs. In another study, 65 subjects who were previously immunized with the HIV-1 Immunogen over a mean period of 4.0 years (range, 1.2-5.4 years) received inoculations at 0 and 6 months. At some point during this 48-week study, 72% of the subjects (47/65) were receiving antiviral drug therapy. The HIV-1 DNA load in CD4 cells and CD4 percentage were found to be stable over the 48-week period. Delayed-type hypersensitivity to HIV-1 antigens increased after two inoculations with the HIV-1 Immunogen. In these two trials, no serious treatment-related adverse events were documented in the subjects. The two studies presented herein are the first to suggest that an immune-based therapy such as the HIV-1 Immunogen can be combined safely with antiviral drugs, supporting further study to evaluate the clinical utility of this approach.

AIDS Vaccines↗
PubMed1996

Osteoma of the internal auditory canal: case report.

A 42-year-old man with normal hearing presented with a long history of vertigo and tinnitus. CT scan showed large osteomata of the internal auditory canal. Magnetic resonance imaging (MRI) was normal. The osteomata were removed surgically via the retrosigmoid approach and examined histologically. His symptoms were abolished. As MRI has become the gold standard in the search for small acoustic tumours it is likely that symptomatic bony lesions, rare though they are, will be missed if MRI is the sole imaging modality. A review of the literature is included.

Adult↗
PubMed1996

Post-stapedectomy reparative granuloma: a misnomer.

The pathophysiology of so-called 'reparative granuloma' occurring after stapedectomy has not been determined and universally accepted management of this rare complication has not yet been established. A case is presented in which a mass developed in the middle ear after the use of a fat/wire prosthesis in a stapedectomy. Histological assessment revealed nonspecific granulation tissue and fat necrosis. It is suggested that 'reparative granuloma' is a misnomer as there is no microscopic evidence in this case nor in the literature of granulomatous formation. The condition may follow stapedectomy or stapedotomy. Furthermore, the name leads to confusion with a different condition, giant cell reparative granuloma, which involves the jaws and rarely the temporal bone. An alternative name, 'Stapes surgery induced granulation tissue' (SSIG) is therefore suggested for this condition.

Adult↗
PubMed1996

Clamp loading, unloading and intrinsic stability of the PCNA, beta and gp45 sliding clamps of human, E. coli and T4 replicases.

BACKGROUND: The high speed and processivity of replicative DNA polymerases reside in a processivity factor which has been shown to be a ring-shaped protein. This protein ("sliding clamp') encircles DNA and tethers the catalytic unit to the template. Although in eukaryotic, prokaryotic and bacteriophage-T4 systems, the processivity factors are ring-shaped, they assume different oligomeric states. The Escherichia coli clamp (the beta subunit) is active as a dimer while the eukaryotic and T4 phage clamps (PCNA and gp45, respectively) are active as trimers. The clamp can not assemble itself on DNA. Instead, a protein complex known as a clamp loader utilizes ATP to assemble the ring around the primer-template. This study compares properties of the human PCNA clamp with those of E. coli and T4 phage. RESULTS: The PCNA ring is a stable trimer down to a concentration below 100 nM (Kd approximately 21 nM). On DNA, the PCNA clamp slides freely and dissociates from DNA slowly (t1/2 approximately 24 min). beta is more stable in solution (Kd < 60 PM) and on DNA (t1/2 approximately 1 h) than PCNA which may be explained by its simpler oligomeric state. The T4 gp45 clamp is a much less stable trimer than PCNA (Kd approximately 250 nM) and requires association with the polymerase to stabilize it on DNA as observed previously. The consequence of this cooperation between clamp and polymerase is that upon finishing a template and dissociation of the polymerase from DNA, the gp45 clamp spontaneously dissociates from DNA without assistance. However, the greater stability of the PCNA and beta clamps on DNA necessitates an active process for their removal. The clamp loaders (RFC and gamma complex) were also capable of unloading their respective clamps from DNA in the presence of ATP. CONCLUSIONS: The stability of the different clamps in solution correlates with their stability on DNA. Thus, the low stability of the T4 clamp explains the inability to isolate gp45 on DNA. The stability of the PCNA and beta clamps predicts they will require an unloading factor to recycle them on and off DNA during replication. The clamp loaders of PCNA and beta double as clamp unloaders presumably for the purpose of clamp recycling.

Bacteriophage T4↗
PubMed1996

Stimulation of human peripheral blood mononuclear cells with live Mycobacterium bovis BCG activates cytolytic CD8+ T cells in vitro.

Experimental data have shown that Mycobacterium tuberculosis can survive within the host cell and in doing so may release secreted antigen into the endogenous antigen-processing pathway. If mycobacterial antigen can gain access to MHC class I molecules then CD8+ T cells may play a role in host defence against M. tuberculosis infection. To identify whether there is a role for the CD8+ T cell in mycobacterial infection we have stimulated peripheral blood mononuclear cells (PBMC) from bacillus Calmette-Guérin (BCG) vaccinated individuals with live M. bovis BCG. The activation state of the T cells was established by staining for the interleukin-2 (IL-2) receptor (CD25), HLA-DR or the transferrin receptor (CD71). Using FACScan analysis we have shown that, in vitro, live M. bovis BCG activates significantly more CD8+ T cells in comparison to the soluble antigen purified protein derivative (PPD). In addition, live M. bovis BCG activates more CD8+ T cells than a non-viable preparation of the same M. bovis BCG following irradiation. The function of the activated CD8+ T cells was addressed using positively selected cells in a cytotoxic T-cell assay. CD8+ T cells isolated from a 7-day M. bovis BCG-stimulated PBMC culture were shown to be cytolytic against target cells infected with live M. bovis BCG, dead M. bovis BCG, and to a lesser extent, PPD. These results suggest that CD8+ T cells may be activated by stimulation with live mycobacteria, and that this subset can play a cytolytic role in the immune response to mycobacterial infections.

Antigens, Bacterial↗
PubMed1996

A histopathologic review of temporal bone exostoses and osteomata.

It is widely accepted that external auditory canal exostoses and osteomata are separate clinical entities that differ in their gross appearance. Disagreement still exists as to whether external auditory canal exostoses and osteomata should be considered similar or separate histopathologic entities. A chart review was performed of all patients who had external auditory canal exostoses or temporal bone osteomata excised from January 1991 to November 1994 at St. Vincent's Hospital, Sydney. A histologic examination was undertaken with a blind study method of the patients whose pathological specimens were suitable for assessment. Eight exostoses and five osteomata were available for examination. The results of this study demonstrate that exostoses and osteomata of the external auditory canal cannot be reliably differentiated by routine histopathological examination.

Bone Neoplasms↗