Cloning and gastrointestinal expression of rat hephaestin: Relationship to other iron transport proteins

The membrane-bound ceruloplasmin homolog hephaestin plays a critical role in intestinal iron absorption. The aims of this study were to clone the rat hephaestin gene and to examine its expression in the gastrointestinal tract in relation to other genes encoding iron transport proteins. The rat hephaestin gene was isolated from intestinal mRNA and was found to encode a protein 96% identical to mouse hephaestin. Analysis by ribonuclease protection assay and Western blotting showed that hephaestin was expressed at high levels throughout the small intestine and colon. Immunofluorescence localized the hephaestin protein to the mature villus enterocytes with little or no expression in the crypts. Variations in iron status had a small but nonsignificant effect on hephaestin expression in the duodenum. The high sequence conservation between rat and mouse hephaestin is consistent with this protein playing a central role in intestinal iron absorption, although its precise function remains to be determined.

Observations on catalysis by hammerhead ribozymes are consistent with a two-divalent-metal-ion mechanism

Significant cleavage by hammerhead ribozymes requires activation by divalent metal ions. Several models have been proposed to account for the influence of metal ions on hammerhead activity. A number of recent papers have presented data that have been interpreted as supporting a one-metal-hydroxide-ion mechanism. In addition, a solvent deuterium isotope effect has been taken as evidence against a proton transfer in the rate-limiting step of the cleavage reaction. We propose that these data are more easily explained by a two-metal-ion mechanism that does not involve a metal hydroxide, but does involve a proton transfer in the rate-limiting step.

Modulation of the SHBG signalling axis

Sex hormone-binding globulin (SHBG) is a homodimeric plasma glycoprotein that is the major sex steroid carrier-protein in the bloodstream and functions also as a key regulator of steroid bioavailability within target tissues, such as the prostate. Additionally, SHBG binds to prostatic cell membranes via the putative and unidentified SHBG receptor (RSHBG), activating a signal transduction pathway implicated in stimulating both proliferation and expression of prostate specific antigen (PSA) in prostate cell lines in vitro. A yeast-two hybrid assay suggested an interaction between SHBG and kallikrein-related protease (KLK) 4, which is a serine protease implicated in the progression of prostate cancer. The potential interaction between these two proteins was investigated in this PhD thesis to determine whether SHBG is a proteolytic substrate of KLK4 and other members of the KLK family including KLK3/PSA, KLK7 and KLK14. Furthermore, the effects from SHBG proteolytic degradation on SHBG-regulated steroid bioavailability and the activation of the putative RSHBG signal transduction pathway were examined in the LNCaP prostate cancer cell line. SHBG was found to be a proteolytic substrate of the trypsin-like KLK4 and KLK14 in vitro, yielding several proteolysis fragments. Both chymotrypsin-like PSA and KLK7 displayed insignificant proteolytic activity against SHBG. The kinetic parameters of SHBG proteolysis by KLK4 and KLK14 demonstrate a strong enzyme-substrate binding capacity, possessing a Km of 1.2 ± 0.7 µM and 2.1 ± 0.6 µM respectively. The catalytic efficiencies (kcat/Km) of KLK4 and KLK14 proteolysis of SHBG were 1.6 x 104 M-1s-1 and 3.8 x 104 M-1s-1 respectively, which were comparable to parameters previously reported for peptide substrates. N-terminal sequencing of the fragments revealed cleavage near the junction of the N- and C-terminal laminin globulin-like (G-like) domains of SHBG, resulting in the division of the two globulins and ultimately the full degradation of these fragments by KLK4 and KLK14 over time. Proteolytic fragments that may retain steroid binding were rapidly degraded by both proteases, while fragments containing residues beyond the steroid binding pocket were less degraded over the same period of time. Degradation of SHBG was inhibited by the divalent metal cations calcium and zinc for KLK4, and calcium, zinc and magnesium for KLK14. The human secreted serine protease inhibitors (serpins), α1-antitrypsin and α2-antiplasmin, inhibited KLK4 and KLK14 proteolysis of SHBG; α1-antichymotrypsin inhibited KLK4 but not KLK14 activity. The inhibition by these serpins was comparable and in some cases more effective than general trypsin protease inhibitors such as aprotinin and phenylmethanesulfonyl fluoride (PMSF). The binding of 5α-dihydrotestosterone (DHT) to SHBG modulated interactions with KLK4 and KLK14. Steroid-free SHBG was more readily digested by both enzymes than DHT-bound SHBG. Moreover, a binding interaction exists between SHBG and pro-KLK4 and pro-KLK14, with DHT strengthening the binding to pro-KLK4 only. The inhibition of androgen uptake by cultured prostate cancer cells, mediated by SHBG steroid-binding, was examined to assess whether SHBG proteolysis by KLK4 and KLK14 modulated this process. Proteolytic digestion eliminated the ability of SHBG to inhibit the uptake of DHT from conditioned media into LNCaP cells. Therefore, the proteolysis of SHBG by KLK4 and KLK14 increased steroid bioavailability in vitro, leading to an increased uptake of androgens by prostate cancer cells. Interestingly, different transcriptional responses of PSA and KLK2, which are androgen-regulated genes, to DHT-bounsd SHBG treatment were observed between low and high passage number LNCaP cells (lpLNCaP and hpLNCaP respectively). HpLNCaP cells treated with DHT-bound SHBG demonstrated a significant synergistic upregulation of PSA and KLK2 above DHT or SHBG treatment alone, which is similar to previously reported downstream responses from RSHBG-mediated signaling activation. As this result was not seen in lpLNCaP cells, only hpLNCaP cells were further investigated to examine the modulation of potential RSHBG activity by KLK4 and KLK14 proteolysis of SHBG. Contrary to reported results, no increase in intracellular cAMP was observed in hpLNCaP cells when treated with SHBG in the presence and absence of either DHT or estradiol. As a result, the modulation of RSHBG-mediated signaling activation could not be determined. Finally, the identification of the RSHBG from both breast (MCF-7) and prostate cancer (LNCaP) cell lines was attempted. Fluorescently labeled peptides corresponding to the putative receptor binding domain (RBD) of SHBG were shown to be internalized by MCF-7 cells. Crosslinking of the RBD peptide to the cell surfaces of both MCF-7 and LNCaP cells, demonstrated the interaction of the peptide with several targets. These targets were then captured using RBD peptides synthesized onto a hydrophilic scaffold and analysed by mass spectrometry. The samples captured by the RBD peptide returned statistically significantly matches for cytokeratin 8, 18 and 19 as well as microtubule-actin crosslinking factor 1, which may indicate a novel interaction between SHBG and these proteins, but ultimately failed to detect a membrane receptor potentially responsible for the putative RSHBG-mediated signaling. This PhD project has reported the proteolytic processing of SHBG by two members of the kallikrein family, KLK4 and KLK14. The effect of SHBG proteolysis by KLK4 and KLK14 on RSHBG-mediated signaling activation was unable to be determined as the reported signal transduction pathway was not activated after treatment with SHBG, in combination with either DHT or estradiol. However, the digestion of SHBG by these two proteases positively regulated androgen bioavailability to prostate cancer cells in vitro. The increased uptake of androgens is deleterious in prostate cancer due to the promotion of proliferation, metastasis, invasion and the inhibition of apoptosis. The increased bioavailability of androgens, from SHBG proteolysis by KLK4 and KLK14, may therefore promote both carcinogenesis and progression of prostate cancer. Finally, this information may contribute to the development of therapeutic treatment strategies for prostate cancer by inhibiting the proteolysis of SHBG, by KLK4 and KLK14, to prevent the increased uptake of androgens by hormone-dependent cancerous tissues.

Inactivation and structural changes of E. Cloacae and B. Subtilis Endospores during IR laser water treatment

IR radiation has been studied for micro-organism inactivation of bacterial spores on metal substrates [1] and on metal and paper substrates [2]. A near-point near infrared laser water treatment apparatus for use in dental hand-pieces was also developed [3]. To date water sterilisation research using a mid-IR laser technique is very rare. According to the World Health Organisation [4], examinations for faecal indicator bacteria remain the most sensitive and specific way of assessing the hygienic quality of water. Bacteria that fall into this group are E. coli, other coliform bacteria (including E. cloacae) and to a lesser extent, faecal streptococci [5]. Protozoan cysts from organisms which cause giardiasis are the most frequently identified cause of waterborne diseases in developed countries [6,7]. The use of aerobic bacterial endospores to monitor the efficiency of various water treatments has been shown to provide a reliable and simple indicator of overall performance of water treatment[8,9].The efficacy of IR radiation for water disinfection compared to UV treatment has been further investigated in the present study. In addition FTIR spectroscopy in conjunction with Principle Component Analysis was used to characterise structural changes within the bacterial cells and endospores following IR laser treatment. Changes in carbohydrate content of E. cloacae following IR laser treatment were observed.

The biology of the glutamatergic system and potential role in migraine

Migraine is a common genetically linked neurovascular disorder. Approximately ~12% of the Caucasian population are affected including 18% of adult women and 6% of adult men (1, 2). A notable female bias is observed in migraine prevalence studies with females affected ~3 times more than males and is credited to differences in hormone levels arising from reproductive achievements. Migraine is extremely debilitating with wide-ranging socioeconomic impact significantly affecting people's health and quality of life. A number of neurotransmitter systems have been implicated in migraine, the most studied include the serotonergic and dopaminergic systems. Extensive genetic research has been carried out to identify genetic variants that may alter the activity of a number of genes involved in synthesis and transport of neurotransmitters of these systems. The biology of the Glutamatergic system in migraine is the least studied however there is mounting evidence that its constituents could contribute to migraine. The discovery of antagonists that selectively block glutamate receptors has enabled studies on the physiologic role of glutamate, on one hand, and opened new perspectives pertaining to the potential therapeutic applications of glutamate receptor antagonists in diverse neurologic diseases. In this brief review, we discuss the biology of the Glutamatergic system in migraine outlining recent findings that support a role for altered Glutamatergic neurotransmission from biochemical and genetic studies in the manifestation of migraine and the implications of this on migraine treatment.

Prolonged hyperinsulinemia affects metabolic signal transduction markers in a tissue specific manner

Insulin dysregulation is common in horses although the mechanisms of metabolic dysfunction are poorly understood. We hypothesized that insulin signaling in striated (cardiac and skeletal) muscle and lamellae may be mediated through different receptors as a result of receptor content, and that transcriptional regulation of downstream signal transduction and glucose transport may also differ between tissues sites during hyperinsulinemia. Archived samples from horses treated with a prolonged insulin infusion or a balanced electrolyte solution were used. All treated horses developed marked hyperinsulinemia and clinical laminitis. Protein expression was compared across tissues for the insulin receptor and insulin-like growth factor 1 receptor (IGF-1R) by immunoblotting. Gene expression of metabolic insulin-signaling markers (insulin receptor substrate 1, Akt2, and glycogen synthase kinase 3 beta [GSK-3β]) and glucose transport (basal glucose transporter 1 and insulin-sensitive glucose transporter 4) was evaluated using real-time reverse transcription polymerase chain reaction. Lamellar tissue contained significantly more IGF-1R protein than skeletal muscle, indicating the potential significance of IGF-1R signaling for this tissue. Gene expression of the selected markers of insulin signaling and glucose transport in skeletal muscle and lamellar tissues was unaffected by prolonged hyperinsulinemia. In contrast, the significant upregulation of Akt2, GSK-3β, GLUT1, and GLUT4 gene expression in cardiac tissue suggested that the prolonged hyperinsulinemia induced an increase in insulin sensitivity and a transcriptional activation of glucose transport. Responses to insulin are tissue-specific, and extrapolation of data across tissue sites is inappropriate.

Organic–inorganic bismuth (III)-based material: A lead-free, air-stable and solution-processable light-absorber beyond organolead perovskites

Methylammonium bismuth (III) iodide single crystals and films have been developed and investigated. We have further presented the first demonstration of using this organic–inorganic bismuth-based material to replace lead/tin-based perovskite materials in solution-processable solar cells. The organic–inorganic bismuth-based material has advantages of non-toxicity, ambient stability, and low-temperature solution-processability, which provides a promising solution to address the toxicity and stability challenges in organolead- and organotin-based perovskite solar cells. We also demonstrated that trivalent metal cation-based organic–inorganic hybrid materials can exhibit photovoltaic effect, which may inspire more research work on developing and applying organic-inorganic hybrid materials beyond divalent metal cations (Pb (II) and Sn (II)) for solar energy applications.

Metal hexaborides with Sc, Ti or Mn

Comparison of well-determined single crystal data for stoichiometric, or near-stoichiometric, metal hexaborides con-firm previously identified lattice parameter trends using powder diffraction. Trends for both divalent and trivalent forms suggest that potential new forms for synthesis include Sc and Mn hexaborides. Density Functional Theory (DFT) calculations for KB6, CaB6, YB6, LaB6, boron octahedral clusters and Sc and Mn forms, show that the shapes of bonding orbitals are defined by the boron framework. Inclusion of metal into the boron framework induces a reduction in energy ranging from 1 eV to 6 eV increasing with ionic charge. For metals with d1 character, such a shift in energy brings a doubly degenerate band section along the G-M reciprocal space direction within the conduction bands tangential to the Fermi surface. ScB6 band structure and density of states calculations show directional and gap characteristics similar to those of YB6 and LaB6. These calculations for ScB6 suggest it may be possible to realize superconductivity in this compound if synthesized.

Metal artifacts from titanium and steel screws in CT, 1.5T and 3T MR images of the tibial Pilon: A quantitative assessment in 3D

Radiographs are commonly used to assess articular reduction of the distal tibia (pilon) fractures postoperatively, but may reveal malreductions inaccurately. While Magnetic Resonance Imaging (MRI) and Computed Tomography (CT) are potential 3D alternatives they generate metal-related artifacts. This study aims to quantify the artifact size from orthopaedic screws using CT, 1.5T and 3T MRI data. Three screws were inserted into one intact human cadaver ankle specimen proximal to and along the distal articular surface, then CT, 1.5T and 3T MRI scanned. Four types of screws were investigated: titanium alloy (TA), stainless steel (SS) (Ø = 3.5 mm), cannulated TA (CTA) and cannulated SS (CSS)(Ø = 4.0 mm, Ø empty core = 2.6 mm). 3D artifact models were reconstructed using adaptive thresholding. The artifact size was measured by calculating the perpendicular distance from the central screw axis to the boundary of the artifact in four anatomical directions with respect to the distal tibia. The artifact sizes (in the order of TA, SS, CTA and CSS) from CT were 2.0 mm, 2.6 mm, 1.6 mm and 2.0 mm; from 1.5T MRI they were 3.7 mm, 10.9 mm, 2.9 mm, and 9 mm; and 3T MRI they were 4.4 mm, 15.3 mm, 3.8 mm, and 11.6 mm respectively. Therefore, CT can be used as long as the screws are at a safe distance of about 2 mm from the articular surface. MRI can be used if the screws are at least 3 mm away from the articular surface except SS and CSS. Artifacts from steel screws were too large thus obstructed the pilon from being visualised in MRI. Significant differences (P < 0.05) were found in the size of artifacts between all imaging modalities, screw types and material types, except 1.5T versus 3T MRI for the SS screws (P = 0.063). CTA screws near the joint surface can improve postoperative assessment in CT and MRI. MRI presents a favourable non-ionising alternative when using titanium hardware. Since these factors may influence the quality of postoperative assessment, potential improvements in operative techniques should be considered.

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.

Defining the flammability of cylindrical metal rods through characterization of the thermal effects of the ignition promoter

All relevant international standards for determining if a metallic rod is flammable in oxygen utilize some form of “promoted ignition” test. In this test, for a given pressure, an overwhelming ignition source is coupled to the end of the test sample and the designation flammable or nonflammable is based upon the amount burned, that is, a burn criteria. It is documented that (1) the initial temperature of the test sample affects the burning of the test sample both (a) in regards to the pressure at which the sample will support burning (threshold pressure) and (b) the rate at which the sample is melted (regression rate of the melting interface); and, (2) the igniter used affects the test sample by heating it adjacent to the igniter as ignition occurs. Together, these facts make it necessary to ensure, if a metallic material is to be considered flammable at the conditions tested, that the burn criteria will exclude any region of the test sample that may have undergone preheating during the ignition process. A two-dimensional theoretical model was developed to describe the transient heat transfer occurring and resultant temperatures produced within this system. Several metals (copper, aluminum, iron, and stainless steel) and ignition promoters (magnesium, aluminum, and Pyrofuze®) were evaluated for a range of oxygen pressures between 0.69 MPa (100 psia) and 34.5 MPa (5,000 psia). A MATLAB® program was utilized to solve the developed model that was validated against (1) a published solution for a similar system and (2) against experimental data obtained during actual tests at the National Aeronautics and Space Administration White Sands Test Facility. The validated model successfully predicts temperatures within the test samples with agreement between model and experiment increasing as test pressure increases and/or distance from the promoter increases. Oxygen pressure and test sample thermal diffusivity were shown to have the largest effect on the results. In all cases evaluated, there is no significant preheating (above about 38°C/100°F) occurring at distances greater than 30 mm (1.18 in.) during the time the ignition source is attached to the test sample. This validates a distance of 30 mm (1.18 in.) above the ignition promoter as a burn length upon which a definition of flammable can be based for inclusion in relevant international standards (that is, burning past this length will always be independent of the ignition event for the ignition promoters considered here. KEYWORDS: promoted ignition, metal combustion, heat conduction, thin fin, promoted combustion, burn length, burn criteria, flammability, igniter effects, heat affected zone.

Tris[4,4,4-trifluoro-1-(2-thienyl)butane-1,3-dionato]aluminium(III)-tris[4,4,4-trifluoro-1-(2-thienyl)butane-1,3-dionato]iron(III) (3/1)

In the title compound, [Al(C8H4F3O2S)3]3[Fe(C8H4F3O2S)3], the metal centre is statistically occupied by AlIII and FeIII cations in a 3:1 ratio. The metal centre is within an octahedral O6 donor set defined by three chelating substituted acetoacetonate anions. The ligands are arranged around the periphery of the molecule with a mer geometry of the S atoms.

High-performance ceramic membranes with a separation layer of metal oxide nanofibers

In conventional fabrication of ceramic separation membranes, the particulate sols are applied onto porous supports. Major structural deficiencies under this approach are pin-holes and cracks, and the dramatic losses of flux when pore sizes are reduced to enhance selectivity. We have overcome these structural deficiencies by constructing hierarchically structured separation layer on a porous substrate using lager titanate nanofibers and smaller boehmite nanofibers. This yields a radical change in membrane texture. The resulting membranes effectively filter out species larger than 60 nm at flow rates orders of magnitude greater than conventional membranes. This reveals a new direction in membrane fabrication.

Ceramic membranes for separation of proteins and DNA by in situ growth of alumina nanofibres with a nanomesh of metal oxide nanofibres

Ceramic membranes were fabricated by in situ synthesis of alumina nanofibres in the pores of an alumina support as a separation layer, and exhibited a high permeation selectivity for bovine serum albumin relative to bovine hemoglobin (over 60 times) and can effectively retain DNA molecules at high fluxes.