Relationship of Omega-3 Fatty Acids on C-Reactive Protein and Homocysteine in Haitian and African Americans with and without Type 2 Diabetes

Background: Omega-3 fatty acids (n-3) may be protective of cardiovascular risk factors for vulnerable populations. The purpose of this study was to assess the association between n-3 with, C-reactive protein (CRP), and homocysteine (HCY) in Black minorities with and without type 2 diabetes. Methods: A cross-sectional study was conducted with 406 participants: Haitian Americans (HA): n=238. African Americans (AA): n=172. Participants were recruited from a randomly generated mailing lists, local diabetes educators, community health practitioners and advertisements from 2008-2010. Sociodemographics and anthropometrics were collected and used to adjust analyses. All dietary variables were collected using the semi-quantitative food frequency questionnaire (FFQ) and used to quantify vitamin components. Blood was collected to measure CVD risk factors (blood lipids, HCY, and CRP). Results: African Americans had higher waist circumferences and C-reactive protein and consumed more calories as compared to Haitian Americans. Omega 3 fatty acid intake per calorie did not differ between these ethnicities, yet African Americans with low n-3 intake were three times more likely to have high C-reactive protein as compared to their counterparts [OR=3. 32 (1. 11, 9. 26) p=0.031]. Although homocysteine did not differ by ethnicity, African Americans with low omega 3 intake (<1 g/day) were four times as likely to have high homocysteine (>12 mg/L) as compared to their counterparts, adjusting for confounders [OR=4.63 (1.59, 12.0) p=0.004]. Consumption of n-3 by diabetes status was not associated with C-reactive protein or homocysteine levels. Conclusions: Consumption of n-3 may be protective of cardiovascular risk factors such as C-r

Obesity and Serum High Sensitivity C-Reactive Protein Levels Among Elderly Turkish Immigrants in the Netherlands with Type 2 Diabetes

The study examined the associations of anthropometric measures of obesity with high sensitivity C-reactive protein (hs-CRP) levels in Turkish immigrants with type 2 diabetes (T2D) living in the Netherlands. A total of 110 participants, physician-diagnosed with T2D, aged 30 years and older were recruited from multiple sources from The Hague, Netherlands. Serum hs-CRP levels were measured with immunoturbidimetric assay. Glycated hemoglobin (A1C) was determined by high-pressure liquid chromatography. Measures of obesity: body weight, body mass index (BMI), waist circumference (WC), hip circumference (HC), waist-to-hip ratio (WHR), and waist-to-height ratio (WHtR) were determined. Statistical analysis included descriptive statistics, Pearson’s correlations and multiple linear regressions (MLR) stratified by gender. Hs-CRP was log transformed to achieve normality. Subjects with hs-CRP levels >10 mg/L (n = 17) were excluded from the analysis. Females had a higher BMI (p = 0.007), HC (p < 0.001), and WHtR (p = 0.011) as compared to males. Conversely, males had a higher weight (p = 0.007), and WHR (p < 0.001) than females. MLR showed that after controlling for covariates, log hs-CRP was positively associated with BMI (B = 0.039, SE = 0.019, β = 0.287, p < 0.05), WC (B = 0.025, SE = 0.011, β = 0.332, p < 0.05) and WHtR (B = 4.015, SE = 1.464, β = 0.376, p < 0.01) in females only. Gender-specific associations between obesity measures and hs-CRP level need to be further investigated in the Turkish immigrant population. Hs-CRP assessment may be added as a standard of care for T2D treatment within this population.

The conceptus induces a switch in protein expression and activities of superoxide dismutase 1 and 2 in the sheep endometrium during early pregnancy

Acknowledgements We thank Philippe Bolifraud (INRA, France), Krawiec Angele, Sandra Grange, Laurence Puillet-Anselme (CHU Grenoble, France) and Margaret Fraser (Aberdeen, UK) for their expert technical assistance. The authors also thank the staff of the sheep sheds of Jouy-en-Josas (INRA, France). The authors would also like to thank the anonymous reviewers for their close examination of this article and their useful comments.

Calcium/Calmodulin-Dependent Protein Kinase Kinase 2 (CaMKK2) Regulates Dendritic Cells and Myeloid Derived Suppressor Cells Development in the Lymphoma Microenvironment

Calcium (Ca2+) is a known important second messenger. Calcium/Calmodulin (CaM) dependent protein kinase kinase 2 (CaMKK2) is a crucial kinase in the calcium signaling cascade. Activated by Ca2+/CaM, CaMKK2 can phosphorylate other CaM kinases and AMP-activated protein kinase (AMPK) to regulate cell differentiation, energy balance, metabolism and inflammation. Outside of the brain, CaMKK2 can only be detected in hematopoietic stem cells and progenitors, and in the subsets of mature myeloid cells. CaMKK2 has been noted to facilitate tumor cell proliferation in prostate cancer, breast cancer, and hepatic cancer. However, whethter CaMKK2 impacts the tumor microenvironment especially in hematopoietic malignancies remains unknown. Due to the relevance of myeloid cells in tumor growth, we hypothesized that CaMKK2 has a critical role in the tumor microenvironment, and tested this hyopothesis in murine models of hematological and solid cancer malignancies.

We found that CaMKK2 ablation in the host suppressed the growth of E.G7 murine lymphoma, Vk*Myc myeloma and E0771 mammary cancer. The selective ablation of CaMKK2 in myeloid cells was sufficient to restrain tumor growth, of which could be reversed by CD8 cell depletion. In the lymphoma microenvironment, ablating CaMKK2 generated less myeloid-derived suppressor cells (MDSCs) in vitro and in vivo. Mechanistically, CaMKK2 deficient dendritic cells showed higher Major Histocompatibility Class II (MHC II) and costimulatory factor expression, higher chemokine and IL-12 secretion when stimulated by LPS, and have higher potent in stimulating T-cell activation. AMPK, an anti-inflammatory kinase, was found as the relevant downstream target of CaMKK2 in dendritic cells. Treatment with CaMKK2 selective inhibitor STO-609 efficiently suppressed E.G7 and E0771 tumor growth, and reshaped the tumor microenvironment by attracting more immunogenic myeloid cells and infiltrated T cells.

In conclusion, we demonstrate that CaMKK2 expressed in myeloid cells is an important checkpoint in tumor microenvironment. Ablating CaMKK2 suppresses lymphoma growth by promoting myeloid cells development thereby decreasing MDSCs while enhancing the anti-tumor immune response. CaMKK2 inhibition is an innovative strategy for cancer therapy through reprogramming the tumor microenvironment.

Molecular mechanism of activation-triggered subunit exchange in Ca(2+)/calmodulin-dependent protein kinase II.

Activation triggers the exchange of subunits in Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), an oligomeric enzyme that is critical for learning, memory, and cardiac function. The mechanism by which subunit exchange occurs remains elusive. We show that the human CaMKII holoenzyme exists in dodecameric and tetradecameric forms, and that the calmodulin (CaM)-binding element of CaMKII can bind to the hub of the holoenzyme and destabilize it to release dimers. The structures of CaMKII from two distantly diverged organisms suggest that the CaM-binding element of activated CaMKII acts as a wedge by docking at intersubunit interfaces in the hub. This converts the hub into a spiral form that can release or gain CaMKII dimers. Our data reveal a three-way competition for the CaM-binding element, whereby phosphorylation biases it towards the hub interface, away from the kinase domain and calmodulin, thus unlocking the ability of activated CaMKII holoenzymes to exchange dimers with unactivated ones.

Molecular mechanism of activation-triggered subunit exchange in Ca(2+)/calmodulin-dependent protein kinase II.

Activation triggers the exchange of subunits in Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), an oligomeric enzyme that is critical for learning, memory, and cardiac function. The mechanism by which subunit exchange occurs remains elusive. We show that the human CaMKII holoenzyme exists in dodecameric and tetradecameric forms, and that the calmodulin (CaM)-binding element of CaMKII can bind to the hub of the holoenzyme and destabilize it to release dimers. The structures of CaMKII from two distantly diverged organisms suggest that the CaM-binding element of activated CaMKII acts as a wedge by docking at intersubunit interfaces in the hub. This converts the hub into a spiral form that can release or gain CaMKII dimers. Our data reveal a three-way competition for the CaM-binding element, whereby phosphorylation biases it towards the hub interface, away from the kinase domain and calmodulin, thus unlocking the ability of activated CaMKII holoenzymes to exchange dimers with unactivated ones.

EGFR interacts with the fusion protein of respiratory syncytial virus strain 2-20 and mediates infection and mucin expression.

Respiratory syncytial virus (RSV) is the major cause of viral lower respiratory tract illness in children. In contrast to the RSV prototypic strain A2, clinical isolate RSV 2-20 induces airway mucin expression in mice, a clinically relevant phenotype dependent on the fusion (F) protein of the RSV strain. Epidermal growth factor receptor (EGFR) plays a role in airway mucin expression in other systems; therefore we hypothesized that the RSV 2-20 F protein stimulates EGFR signaling. Infection of cells with chimeric strains RSV A2-2-20F and A2-2-20GF or over-expression of 2-20 F protein resulted in greater phosphorylation of EGFR than infection with RSV A2 or over-expression of A2 F, respectively. Chemical inhibition of EGFR signaling or knockdown of EGFR resulted in diminished infectivity of RSV A2-2-20F but not RSV A2. Over-expression of EGFR enhanced the fusion activity of 2-20 F protein in trans. EGFR co-immunoprecipitated most efficiently with RSV F proteins derived from “mucogenic” strains. RSV 2-20 F and EGFR co-localized in H292 cells, and A2-2-20GF-induced MUC5AC expression was ablated by EGFR inhibitors in these cells. Treatment of BALB/c mice with the EGFR inhibitor erlotinib significantly reduced the amount of RSV A2-2-20F-induced airway mucin expression. Our results demonstrate that RSV F interacts with EGFR in a strain-specific manner, EGFR is a co-factor for infection, and EGFR plays a role in RSV-induced mucin expression, suggesting EGFR is a potential target for RSV disease.

Exchange protein directly activated by cyclic AMP (EPAC) activation reverses neutrophil dysfunction induced by β2-agonists, corticosteroids, and critical illness

Background Neutrophils play a role in the pathogenesis of asthma, chronic obstructive pulmonary disease, and pulmonary infection. Impaired neutrophil phagocytosis predicts hospital-acquired infection. Despite this, remarkably few neutrophil-specific treatments exist. 

Objectives We sought to identify novel pathways for the restoration of effective neutrophil phagocytosis and to activate such pathways effectively in neutrophils from patients with impaired neutrophil phagocytosis. 

Methods Blood neutrophils were isolated from healthy volunteers and patients with impaired neutrophil function. In healthy neutrophils phagocytic impairment was induced experimentally by using β₂-agonists. Inhibitors and activators of cyclic AMP (cAMP)-dependent pathways were used to assess the influence on neutrophil phagocytosis in vitro. 

Results β₂-Agonists and corticosteroids inhibited neutrophil phagocytosis. Impairment of neutrophil phagocytosis by β₂-agonists was associated with significantly reduced RhoA activity. Inhibition of protein kinase A (PKA) restored phagocytosis and RhoA activity, suggesting that cAMP signals through PKA to drive phagocytic impairment. However, cAMP can signal through effectors other than PKA, such as exchange protein directly activated by cyclic AMP (EPAC). An EPAC-activating analog of cAMP (8CPT-2Me-cAMP) reversed neutrophil dysfunction induced by β₂-agonists or corticosteroids but did not increase RhoA activity. 8CPT-2Me-cAMP reversed phagocytic impairment induced by Rho kinase inhibition but was ineffective in the presence of Rap-1 GTPase inhibitors. 8CPT-2Me-cAMP restored function to neutrophils from patients with known acquired impairment of neutrophil phagocytosis. 

Conclusions EPAC activation consistently reverses clinical and experimental impairment of neutrophil phagocytosis. EPAC signals through Rap-1 and bypasses RhoA. EPAC activation represents a novel potential means by which to reverse impaired neutrophil phagocytosis.

Responses to various protein and energy supplements by steers fed low-quality tropical hay. 2. Effect of stage of maturity of steers

Dose response curves to various supplements were established in two pen-feeding experiments (Exp1 and Exp2) with Bos indicus crossbred steers of two age groups (Young, 10–12 months; Old, 33–36 months) fed low-quality tropical grass hays ad libitum. Diets included supplements based on (Exp1) cottonseed meal (CSM; intake (as fed) 0–10 g/kg liveweight (W).day) and a barley mix (Bar; 0–20 g/kg W.day) and (Exp2) a molasses mix (MUP) and a Bar mix, both fed at 0–20 g/kg W.day. Urea was provided with the Bar mixes and urea/copra meal with the MUP mix. Growth rates of Young steers increased linearly with Bar and MUP supplements but asymptotically with CSM whereas those of Old steers increased asymptotically with all supplement types. With supplement intake expressed on a liveweight basis (g/kg W.day), responses were greater for both steer age groups with CSM compared with Bar (Young, P < 0.001; Old, P < 0.01) and Bar compared with MUP treatments (Young, P < 0.01; Old, P < 0.05). Furthermore, Old steers outperformed their Young counterparts with both CSM (P < 0.05) and Bar (P < 0.001) supplements fed in Exp1 and with Bar and MUP supplements (P < 0.01) fed in Exp2. When supplement intake was expressed in absolute terms (kg/day), growth responses were not different between age groups for different supplements except that Old steers had a higher daily W gain on Bar than their Young counterparts (P < 0.05). Intake of hay (W-corrected) was higher for Young compared with Old steers without supplement but was variably reduced for both steer groups with increasing supplement intake. The results of these experiments have implications for supplement formulation for steers at different stages of maturity grazing low-quality forages.

Elevated hepatocyte growth factor levels in osteoarthritis osteoblasts contribute to their altered response to bone morphogenetic protein-2 and reduced mineralization capacity

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Association between ferritin, high sensitivity c-reactive protein (hsCRP) and relative abundance of Hepcidin mRNA with the risk of type 2 diabetes in obese subjects

Obesity and Type 2 diabetes mellitus share a strong pro-inflammatory profile. It has been observed that iron is a risk factor in the development of type 2 diabetes. The aim of this study was to evaluate the relationship between iron nutritional status and inflammation with the risk of type 2 diabetes development in obese subjects. We studied 30 obese men with type 2 diabetes (OBDM); 30 obese subjects without diabetes (OB) and 30 healthy subjects (Cn). We isolated peripheral mononuclear cells (PMCs) and challenged them with high Fe concentrations. Total mRNA was isolated and relative abundance of TNF-αIL-6 and hepcidin were determined by qPCR. Iron status, biochemical, inflammatory and oxidative stress parameters were also characterized. OBDM and OB patients showed increased hsCRP levels compared to the Cn group. OBDM subjects showed higher levels of ferritin than the Cn group. TNF-α and IL-6 mRNA relative abundances were increased in OBDM PMCs treated with high/Fe. Hepcidin mRNA was increased with basal and high iron concentration. We found that the highest quartile of ferritin was associated with an increased risk of type 2 diabetes when it was adjusted to BMI and HOMA-IR; this association was independent of the inflammatory status. The highest level of hepcidin gene expression also showed a trend of increased risk of diabetes, however it was not significant. Levels of hsCRP over 2 mg/L showed a significant trend of increasing the risk of diabetes. In conclusion, iron may stimulate the expression of pro-inflammatory genes (TNF-α and IL-6), and both hepcidin and ferritin gene expression levels could be a risk factor for the development of type 2 diabetes. Subjects that have an increased cardiovascular risk also have a major risk to develop type 2 diabetes, which is independent of the BMI and insulin resistance state.

Membrane associated transporter protein gene (SLC45A2) and the genetic basis of normal human pigmentation variation

This work is concerned with the genetic basis of normal human pigmentation variation. Specifically, the role of polymorphisms within the solute carrier family 45 member 2 (SLC45A2 or membrane associated transporter protein; MATP) gene were investigated with respect to variation in hair, skin and eye colour ― both between and within populations. SLC45A2 is an important regulator of melanin production and mutations in the gene underly the most recently identified form of oculocutaneous albinism. There is evidence to suggest that non-synonymous polymorphisms in SLC45A2 are associated with normal pigmentation variation between populations. Therefore, the underlying hypothesis of this thesis is that polymorphisms in SLC45A2 will alter the function or regulation of the protein, thereby altering the important role it plays in melanogenesis and providing a mechanism for normal pigmentation variation. In order to investigate the role that SLC45A2 polymorphisms play in human pigmentation variation, a DNA database was established which collected pigmentation phenotypic information and blood samples of more than 700 individuals. This database was used as the foundation for two association studies outlined in this thesis, the first of which involved genotyping two previously-described non-synonymous polymorphisms, p.Glu272Lys and p.Phe374Leu, in four different population groups. For both polymorphisms, allele frequencies were significantly different between population groups and the 272Lys and 374Leu alleles were strongly associated with black hair, brown eyes and olive skin colour in Caucasians. This was the first report to show that SLC45A2 polymorphisms were associated with normal human intra-population pigmentation variation. The second association study involved genotyping several SLC45A2 promoter polymorphisms to determine if they also played a role in pigmentation variation. Firstly, the transcription start site (TSS), and hence putative proximal promoter region, was identified using 5' RNA ligase mediated rapid amplification of cDNA ends (RLM-RACE). Two alternate TSSs were identified and the putative promoter region was screened for novel polymorphisms using denaturing high performance liquid chromatography (dHPLC). A novel duplication (c.–1176_–1174dupAAT) was identified along with other previously described single nucleotide polymorphisms (c.–1721C>G and c.–1169G>A). Strong linkage disequilibrium ensured that all three polymorphisms were associated with skin colour such that the –1721G, +dup and –1169A alleles were associated with olive skin in Caucasians. No linkage disequilibrium was observed between the promoter and coding region polymorphisms, suggesting independent effects. The association analyses were complemented with functional data, showing that the –1721G, +dup and –1169A alleles significantly decreased SLC45A2 transcriptional activity. Based on in silico bioinformatic analysis that showed these alleles remove a microphthalmia-associated transcription factor (MITF) binding site, and that MITF is a known regulator of SLC45A2 (Baxter and Pavan, 2002; Du and Fisher, 2002), it was postulated that SLC45A2 promoter polymorphisms could contribute to the regulation of pigmentation by altering MITF binding affinity. Further characterisation of the SLC45A2 promoter was carried out using luciferase reporter assays to determine the transcriptional activity of different regions of the promoter. Five constructs were designed of increasing length and their promoter activity evaluated. Constitutive promoter activity was observed within the first ~200 bp and promoter activity increased as the construct size increased. The functional impact of the –1721G, +dup and –1169A alleles, which removed a MITF consensus binding site, were assessed using electrophoretic mobility shift assays (EMSA) and expression analysis of genotyped melanoblast and melanocyte cell lines. EMSA results confirmed that the promoter polymorphisms affected DNA-protein binding. Interestingly, however, the protein/s involved were not MITF, or at least MITF was not the protein directly binding to the DNA. In an effort to more thoroughly characterise the functional consequences of SLC45A2 promoter polymorphisms, the mRNA expression levels of SLC45A2 and MITF were determined in melanocyte/melanoblast cell lines. Based on SLC45A2’s role in processing and trafficking TYRP1 from the trans-Golgi network to stage 2 melanosmes, the mRNA expression of TYRP1 was also investigated. Expression results suggested a coordinated expression of pigmentation genes. This thesis has substantially contributed to the field of pigmentation by showing that SLC45A2 polymorphisms not only show allele frequency differences between population groups, but also contribute to normal pigmentation variation within a Caucasian population. In addition, promoter polymorphisms have been shown to have functional consequences for SLC45A2 transcription and the expression of other pigmentation genes. Combined, the data presented in this work supports the notion that SLC45A2 is an important contributor to normal pigmentation variation and should be the target of further research to elucidate its role in determining pigmentation phenotypes. Understanding SLC45A2’s function may lead to the development of therapeutic interventions for oculocutaneous albinism and other disorders of pigmentation. It may also help in our understanding of skin cancer susceptibility and evolutionary adaptation to different UV environments, and contribute to the forensic application of pigmentation phenotype prediction.

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.

The stimulation of healing within a rat calvarial defect by mPCL–TCP/collagen scaffolds loaded with rhBMP-2

Bone morphogenetic proteins (BMPs) have been widely investigated for their clinical use in bone repair and it is known that a suitable carrier matrix to deliver them is essential for optimal bone regeneration within a specific defect site. Fused deposited modeling (FDM) allows for the fabrication of medical grade poly 3-caprolactone/tricalcium phosphate (mPCL–TCP) scaffolds with high reproducibility and tailor designed dimensions. Here we loaded FDM fabricated mPCL–TCP/collagen scaffolds with 5 mg recombinant human (rh)BMP-2 and evaluated bone healing within a rat calvarial critical-sized defect. Using a comprehensive approach, this study assessed the newly regenerated bone employing microcomputed tomography (mCT), histology/histomorphometry, and mechanical assessments. By 15 weeks, mPCL–TCP/collagen/rhBMP-2 defects exhibited complete healing of the calvarium whereas the non- BMP-2-loaded scaffolds showed significant less bone ingrowth, as confirmed by mCT. Histomorphometry revealed significantly increased bone healing amongst the rhBMP-2 groups compared to non-treated scaffolds at 4 and 15 weeks, although the % BV/TV did not indicate complete mineralisation of the entire defect site. Hence, our study confirms that it is important to combine microCt and histomorphometry to be able to study bone regeneration comprehensively in 3D. A significant up-regulation of the osteogenic proteins, type I collagen and osteocalcin, was evident at both time points in rhBMP-2 groups. Although mineral apposition rates at 15 weeks were statistically equivalent amongst treatment groups, microcompression and push-out strengths indicated superior bone quality at 15 weeks for defects treated with mPCL–TCP/collagen/rhBMP-2. Consistently over all modalities, the progression of healing was from empty defect < mPCL–TCP/collagen < mPCL–TCP/collagen/rhBMP-2, providing substantiating data to support the hypothesis that the release of rhBMP-2 from FDM-created mPCL–TCP/collagen scaffolds is a clinically relevant approach to repair and regenerate critically-sized craniofacial bone defects. Crown Copyright 2008 Published by Elsevier Ltd. All rights reserved.

Fluorescence spectrometric study on the interactions of isoprocarb and sodium 2-isopropylphenate with bovine serum albumin

The binding interaction of the pesticide Isoprocarb and its degradation product, sodium 2-isopropylphenate, with bovine serum albumin (BSA) was studied by spectrofluorimetry under simulated physiological conditions. Both Isoprocarb and sodium 2-isopropylphenate quenched the intrinsic fluorescence of BSA. This quenching proceeded via a static mechanism. The thermodynamic parameters (ΔH°, ΔS° and ΔG°) obtained from the fluorescence data measured at two different temperatures showed that the binding of Isoprocarb to BSA involved hydrogen bonds and that of sodium 2-isopropylphenate to BSA involved hydrophobic and electrostatic interactions. Synchronous fluorescence spectroscopy of the interaction of BSA with either Isoprocarb or sodium 2-isopropylphenate showed that the molecular structure of the BSA was changed significantly, which is consistent with the known toxicity of the pesticide, i.e., the protein is denatured. The sodium 2-isopropylphenate, was estimated to be about 4–5 times more toxic than its parent, Isoprocarb. Synchronous fluorescence spectroscopy and the resolution of the three-way excitation–emission fluorescence spectra by the PARAFAC method extracted the relative concentration profiles of BSA, Isoprocab and sodium 2-isopropylphenate as a function of the added sodium 2-isopropylphenate. These profiles showed that the degradation product, sodium 2-isopropylphenate, displaced the pesticide in a competitive reaction with the BSA protein.