Mutants in the Exo I motif of Escherichia coli dnaQ: defective proofreading and inviability due to error catastrophe.

The Escherichia coli dnaQ gene encodes the proofreading 3' exonuclease (epsilon subunit) of DNA polymerase III holoenzyme and is a critical determinant of chromosomal replication fidelity. We constructed by site-specific mutagenesis a mutant, dnaQ926, by changing two conserved amino acid residues (Asp-12-->Ala and Glu-14-->Ala) in the Exo I motif, which, by analogy to other proofreading exonucleases, is essential for the catalytic activity. When residing on a plasmid, dnaQ926 confers a strong, dominant mutator phenotype, suggesting that the protein, although deficient in exonuclease activity, still binds to the polymerase subunit (alpha subunit or dnaE gene product). When dnaQ926 was transferred to the chromosome, replacing the wild-type gene, the cells became inviable. However, viable dnaQ926 strains could be obtained if they contained one of the dnaE alleles previously characterized in our laboratory as antimutator alleles or if it carried a multicopy plasmid containing the E. coli mutL+ gene. These results suggest that loss of proofreading exonuclease activity in dnaQ926 is lethal due to excessive error rates (error catastrophe). Error catastrophe results from both the loss of proofreading and the subsequent saturation of DNA mismatch repair. The probability of lethality by excessive mutation is supported by calculations estimating the number of inactivating mutations in essential genes per chromosome replication.

Neural tube defects and abnormal brain development in F52-deficient mice.

F52 is a myristoylated, alanine-rich substrate for protein kinase C. We have generated F52-deficient mice by the gene targeting technique. These mutant mice manifest severe neural tube defects that are not associated with other complex malformations, a phenotype reminiscent of common human neural tube defects. The neural tube defects observed include both exencephaly and spina bifida, and the phenotype exhibits partial penetrance with about 60% of homozygous embryos developing neural tube defects. Exencephaly is the prominent type of defect and leads to high prenatal lethality. Neural tube defects are observed in a smaller percentage of heterozygous embryos (about 10%). Abnormal brain development and tail formation occur in homozygous mutants and are likely to be secondary to the neural tube defects. Disruption of F52 in mice therefore identifies a gene whose mutation results in isolated neural tube defects and may provide an animal model for common human neural tube defects.

Neonatal lethality associated with respiratory distress in mice lacking cytochrome P450 1A2.

Cytochrome P450 1A2 (CYP1A2) is a constitutively expressed hepatic enzyme that is highly conserved among mammals. This protein is primarily involved in oxidative metabolism of xenobiotics and is capable of metabolically activating numerous procarcinogens including aflatoxin B1, arylamines, heterocyclic amine food mutagens, and polycylic aromatic hydrocarbons. Expression of CYP1A2 is induced after exposure to certain aromatic hydrocarbons (i.e., 2,3,7,8-tetrachlorodibenzo-p-dioxin). Direct evidence for a role of CYP1A2 in any physiological or developmental pathway has not been documented. We now demonstrate that mice homozygous for a targeted mutation in the Cyp1a-2 gene are nonviable. Lethality occurs shortly after birth with symptoms of severe respiratory distress. Mutant neonates display impaired respiratory function associated with histological signs of lung immaturity, lack of air in alveoli at birth, and changes in expression of surfactant apoprotein in alveolar type II cells. The penetrance of the phenotype is not complete (19 mutants survived to adulthood out of 599 mice). Surviving animals, although lacking expression of CYP1A2, appear to be normal and are able to reproduce. These findings establish that CYP1A2 is critical for neonatal survival by influencing the physiology of respiration in neonates, thus offering etiological insights for neonatal respiratory distress syndrome.

Functional analysis of neurotransmission at β2-laminin deficient terminals

beta2-Laminin is important for the formation of neuromuscular junctions in vertebrates. Previously, we have inactivated the gene that encodes for beta2-laminin in mice and observed predominantly prejunctional structural defects. In this study, we have used both intra- and extracellular recording methods to investigate evoked neurotransmission in beta2-laminin-deficient mice, from postnatal day 8 (P8) through to day 18(P18). Our results confirmed that there was a decrease in the frequency of spontaneous release, but no change in the postjunctional response to such release. Analysis of evoked neurotransmission showed an increase in the frequency of stimuli that failed to elicit an evoked postjunctional response in the mutants compared to litter mate controls, resulting in a 50% reduction in mean quantal content at mutant terminals. Compared to littermate controls, beta2-laminin-deficient terminals showed greater synaptic depression when subjected to high frequency stimulation. Furthermore, the paired pulse ratio of the first two stimuli was significantly lower in beta2-laminin mutant terminals. Statistical analysis of the binomial parameters of release showed that the decrease in quantal content was due to a decrease in the number of release sites without any significant change in the average probability of release. This suggestion was supported by the observation of fewer synaptic vesicle protein 2 (SV2)-positive varicosities in beta2-laminin-deficient terminals and by ultrastructural observations showing smaller terminal profiles and increased Schwann cell invasion in beta2-laminin mutants; the differences between beta2-laminin mutants and wild-type mice were the same at both P8 and P18. From these results we conclude that beta2-laminin plays a role in the early structural development of the neuromuscular junction. We also suggest that transmitter release activity may act as a deterrent to Schwarm cell invasion in the absence of beta2-laminin.

The role of growth hormone replacement in a growth hormone deficient patient with underlying cardiomyopathy and severe congestive heart failure

It has been reported that-growth hormone (GH) deficiency induced cardiomyopathy responds to growth hormone replacement therapy. We describe the case of a middle-aged male with cardiomyopathic heart failure and growth hormone deficiency of the adult secondary to surgical panhypopituitarism. We demonstrate clinical and hemodynamic improvement of cardiac function with growth hormone replacement therapy despite underlying structural heart disease. Copyright (C) 2005 by the International Society for Heart and Lung Transplantation.

Sensitivity of antioxidant-deficient yeast to hypochlorite and chlorite

Sodium hypochlorite and sodium chlorite are commonly used as disinfectants, and understanding the mechanisms of microbial resistance to these compounds is of considerable importance. In this study, the role of oxidative stress and antioxidant enzymes in the sensitivity of the yeast Saccharomyces cerevisiae to hypochlorite and chlorite was studied. Yeast mutants lacking Cu-Zn superoxide dismutase, but not mutants deficient in cytoplasmic and peroxisomal catalase, were hypersensitive to the action of both hypochlorite and chlorite. Both compounds depleted cellular glutathione, induced the production of reactive oxygen species and decreased the viability of the cells. The toxicity of hypochlorite and chlorite was abolished by hypoxic and anoxic conditions and ameliorated by thiol antioxidants and ascorbate. The results demonstrated that the action of hypochlorite and chlorite involves the formation of superoxide and peroxide and that SOD1 is protective, probably by limiting the formation of hydroxyl radicals and damage to proteins.

The Fate of Spermatogonial Stem Cells in the Cryptorchid Testes of RXFP2 Deficient Mice

The environmental niche of the spermatogonial stem cell pool is critical to ensure the continued generation of the germ cell population. To study the consequences of an aberrant testicular environment in cryptorchidism we used a mouse model with a deletion of Rxfp2 gene resulting in a high intra-abdominal testicular position. Mutant males were infertile with the gross morphology of the cryptorchid testis progressively deteriorating with age. Few spermatogonia were identifiable in 12 month old cryptorchid testes. Gene expression analysis showed no difference between mutant and control testes at postnatal day 10. In three month old males a decrease in expression of spermatogonial stem cell (SSC) markers Id4, Nanos2, and Ret was shown. The direct counting of ID4+ cells supported a significant decrease of SSCs. In contrast, the expression of Plzf, a marker for undifferentiated and differentiating spermatogonia was not reduced, and the number of PLZF+ cells in the cryptorchid testis was higher in three month old testes, but equal to control in six month old mutants. The PLZF+ cells did not show a higher rate of apoptosis in cryptorchid testis. The expression of the Sertoli cell FGF2 gene required for SSC maintenance was significantly reduced in mutant testis. Based on these findings we propose that the deregulation of somatic and germ cell genes in the cryptorchid testis, directs the SSCs towards the differentiation pathway. This leads to a depletion of the SSC pool and an increase in the number of PLZF+ spermatogonial cells, which too, eventually decreases with the exhaustion of the stem cell pool. Such a dynamic suggests that an early correction of cryptorchidism is critical for the retention of the SSC pool.

Correcting for bias when estimating the cost of hospital-acquired infection : an analysis of lower respiratory tract infections in non-surgical patients

Hospital acquired infections (HAI) are costly but many are avoidable. Evaluating prevention programmes requires data on their costs and benefits. Estimating the actual costs of HAI (a measure of the cost savings due to prevention) is difficult as HAI changes cost by extending patient length of stay, yet, length of stay is a major risk factor for HAI. This endogeneity bias can confound attempts to measure accurately the cost of HAI. We propose a two-stage instrumental variables estimation strategy that explicitly controls for the endogeneity between risk of HAI and length of stay. We find that a 10% reduction in ex ante risk of HAI results in an expected savings of £693 ($US 984).

Distribution of respiratory droplets in enclosed environments under different air distribution methods

Dispersion characteristics of respiratory droplets in indoor environments are of special interest in controlling transmission of airborne diseases. This study adopts an Eulerian method to investigate the spatial concentration distribution and temporal evolution of exhaled and sneezed/coughed droplets within the range of 1.0~10.0μm in an office room with three air distribution methods, i.e. mixing ventilation (MV), displacement ventilation (DV), and under-floor air distribution (UFAD). The diffusion, gravitational settling, and deposition mechanism of particulate matters are well accounted in the one-way coupling Eulerian approach. The simulation results find that exhaled droplets with diameters up to 10.0μm from normal respiration process are uniformly distributed in MV, while they are trapped in the breathing height by thermal stratifications in DV and UFAD, resulting in a high droplet concentration and a high exposure risk to other occupants. Sneezed/coughed droplets are diluted much slower in DV/UFAD than in MV. Low air speed in the breathing zone in DV/UFAD can lead to prolonged residence of droplets in the breathing zone.

Biochemical characterization of Aprataxin, the protein deficient in Ataxia with Oculomotor Apraxia type 1

Neurodegenerative disorders are heterogenous in nature and include a range of ataxias with oculomotor apraxia, which are characterised by a wide variety of neurological and ophthalmological features. This family includes recessive and dominant disorders. A subfamily of autosomal recessive cerebellar ataxias are characterised by defects in the cellular response to DNA damage. These include the well characterised disorders Ataxia-Telangiectasia (A-T) and Ataxia-Telangiectasia Like Disorder (A-TLD) as well as the recently identified diseases Spinocerebellar ataxia with axonal neuropathy Type 1 (SCAN1), Ataxia with Oculomotor Apraxia Type 2 (AOA2), as well as the subject of this thesis, Ataxia with Oculomotor Apraxia Type 1 (AOA1). AOA1 is caused by mutations in the APTX gene, which is located at chromosomal locus 9p13. This gene codes for the 342 amino acid protein Aprataxin. Mutations in APTX cause destabilization of Aprataxin, thus AOA1 is a result of Aprataxin deficiency. Aprataxin has three functional domains, an N-terminal Forkhead Associated (FHA) phosphoprotein interaction domain, a central Histidine Triad (HIT) nucleotide hydrolase domain and a C-terminal C2H2 zinc finger. Aprataxins FHA domain has homology to FHA domain of the DNA repair protein 5’ polynucleotide kinase 3’ phosphatase (PNKP). PNKP interacts with a range of DNA repair proteins via its FHA domain and plays a critical role in processing damaged DNA termini. The presence of this domain with a nucleotide hydrolase domain and a DNA binding motif implicated that Aprataxin may be involved in DNA repair and that AOA1 may be caused by a DNA repair deficit. This was substantiated by the interaction of Aprataxin with proteins involved in the repair of both single and double strand DNA breaks (XRay Cross-Complementing 1, XRCC4 and Poly-ADP Ribose Polymerase-1) and the hypersensitivity of AOA1 patient cell lines to single and double strand break inducing agents. At the commencement of this study little was known about the in vitro and in vivo properties of Aprataxin. Initially this study focused on generation of recombinant Aprataxin proteins to facilitate examination of the in vitro properties of Aprataxin. Using recombinant Aprataxin proteins I found that Aprataxin binds to double stranded DNA. Consistent with a role for Aprataxin as a DNA repair enzyme, this binding is not sequence specific. I also report that the HIT domain of Aprataxin hydrolyses adenosine derivatives and interestingly found that this activity is competitively inhibited by DNA. This provided initial evidence that DNA binds to the HIT domain of Aprataxin. The interaction of DNA with the nucleotide hydrolase domain of Aprataxin provided initial evidence that Aprataxin may be a DNA-processing factor. Following these studies, Aprataxin was found to hydrolyse 5’adenylated DNA, which can be generated by unscheduled ligation at DNA breaks with non-standard termini. I found that cell extracts from AOA1 patients do not have DNA-adenylate hydrolase activity indicating that Aprataxin is the only DNA-adenylate hydrolase in mammalian cells. I further characterised this activity by examining the contribution of the zinc finger and FHA domains to DNA-adenylate hydrolysis by the HIT domain. I found that deletion of the zinc finger ablated the activity of the HIT domain against adenylated DNA, indicating that the zinc finger may be required for the formation of a stable enzyme-substrate complex. Deletion of the FHA domain stimulated DNA-adenylate hydrolysis, which indicated that the activity of the HIT domain may be regulated by the FHA domain. Given that the FHA domain is involved in protein-protein interactions I propose that the activity of Aprataxins HIT domain may be regulated by proteins which interact with its FHA domain. We examined this possibility by measuring the DNA-adenylate hydrolase activity of extracts from cells deficient for the Aprataxin-interacting DNA repair proteins XRCC1 and PARP-1. XRCC1 deficiency did not affect Aprataxin activity but I found that Aprataxin is destabilized in the absence of PARP-1, resulting in a deficiency of DNA-adenylate hydrolase activity in PARP-1 knockout cells. This implies a critical role for PARP-1 in the stabilization of Aprataxin. Conversely I found that PARP-1 is destabilized in the absence of Aprataxin. PARP-1 is a central player in a number of DNA repair mechanisms and this implies that not only do AOA1 cells lack Aprataxin, they may also have defects in PARP-1 dependant cellular functions. Based on this I identified a defect in a PARP-1 dependant DNA repair mechanism in AOA1 cells. Additionally, I identified elevated levels of oxidized DNA in AOA1 cells, which is indicative of a defect in Base Excision Repair (BER). I attribute this to the reduced level of the BER protein Apurinic Endonuclease 1 (APE1) I identified in Aprataxin deficient cells. This study has identified and characterised multiple DNA repair defects in AOA1 cells, indicating that Aprataxin deficiency has far-reaching cellular consequences. Consistent with the literature, I show that Aprataxin is a nuclear protein with nucleoplasmic and nucleolar distribution. Previous studies have shown that Aprataxin interacts with the nucleolar rRNA processing factor nucleolin and that AOA1 cells appear to have a mild defect in rRNA synthesis. Given the nucleolar localization of Aprataxin I examined the protein-protein interactions of Aprataxin and found that Aprataxin interacts with a number of rRNA transcription and processing factors. Based on this and the nucleolar localization of Aprataxin I proposed that Aprataxin may have an alternative role in the nucleolus. I therefore examined the transcriptional activity of Aprataxin deficient cells using nucleotide analogue incorporation. I found that AOA1 cells do not display a defect in basal levels of RNA synthesis, however they display defective transcriptional responses to DNA damage. In summary, this thesis demonstrates that Aprataxin is a DNA repair enzyme responsible for the repair of adenylated DNA termini and that it is required for stabilization of at least two other DNA repair proteins. Thus not only do AOA1 cells have no Aprataxin protein or activity, they have additional deficiencies in PolyADP Ribose Polymerase-1 and Apurinic Endonuclease 1 dependant DNA repair mechanisms. I additionally demonstrate DNA-damage inducible transcriptional defects in AOA1 cells, indicating that Aprataxin deficiency confers a broad range of cellular defects and highlighting the complexity of the cellular response to DNA damage and the multiple defects which result from Aprataxin deficiency. My detailed characterization of the cellular consequences of Aprataxin deficiency provides an important contribution to our understanding of interlinking DNA repair processes.

Deposition of combustion aerosols in the human respiratory tract : comparison of theoretical predictions with experimental data

Total deposition of petrol, diesel and environmental tobacco smoke (ETS) aerosols in the human respiratory tract for nasal breathing conditions was computed for 14 nonsmoking volunteers, considering the specific anatomical and respiratory parameters of each volunteer and the specific size distribution for each inhalation experiment. Theoretical predictions were 34.6% for petrol, 24.0% for diesel, and 18.5% for ETS particles. Compared to the experimental results, predicted deposition values were consistently smaller than the measured data (41.4% for petrol, 29.6% for diesel, and 36.2% for ETS particles). The apparent discrepancy between experimental data on total deposition and modeling results may be reconciled by considering the non-spherical shape of the test aerosols by diameter-dependent dynamic shape factors to account for differences between mobility-equivalent and volume-equivalent or thermodynamic diameters. While the application of dynamic shape factors is able to explain the observed differences for petrol and diesel particles, additional mechanisms may be required for ETS particle deposition, such as the size reduction upon inspiration by evaporation of volatile compounds and/or condensation-induced restructuring, and, possibly, electrical charge effects.

Long-term exposure to gaseous air pollutants and cardio-respiratory mortality in Brisbane, Australia

Air pollution is ranked by the World Health Organisation as one of the top ten contributors to the global burden of disease and injury. Exposure to gaseous air pollutants, even at a low level, has been associated with cardiorespiratory diseases (Vedal, Brauer et al. 2003). Most recent epidemiological studies of air pollution have used time-series analyses to explore the relationship between daily mortality or morbidity and daily ambient air pollution concentrations based on the same day or previous days (Hajat, Armstrong et al. 2007). However, most of the previous studies have examined the association between air pollution and health outcomes using air pollution data from a single monitoring site or average values from a few monitoring sites to represent the whole population of the study area. In fact, for a metropolitan city, ambient air pollution levels may differ significantly among the different areas. There is increasing concern that the relationships between air pollution and mortality may vary with geographical area (Chen, Mengersen et al. 2007). Additionally, some studies have indicated that socio-economic status can act as a confounder when investigating the relation between geographical location and health (Scoggins, Kjellstrom et al. 2004). This study examined the spatial variation in the relationship between long-term exposure to gaseous air pollutants (including nitrogen dioxide (NO2), ozone (O3) and sulphur dioxide (SO2)), and cardiorespiratory mortality in Brisbane, Australia, during the period 1996 - 2004.