The mechanism of maturation of insulin-like growth factor-1

This study, to elucidate the role of des(1-3)IGF-I in the maturation of IGF-I,used two strategies. The first was to detect the presence of enzymes in tissues, which would act on IGF-I to produce des(1-3)IGF-I, and the second was to detect the potential products of such enzymic activity, namely Gly-Pro-Glu(GPE), Gly-Pro(GP) and des(l- 3)IGF-I. No neutral tripeptidyl peptidase (TPP II), which would release the tripeptide GPE from IGF-I, was detected in brain, urine nor in red or white blood cells. The TPPlike activity which was detected, was attributed to a combined action of a dipeptidyl peptidase (DPP N) and an aminopeptidase (AP A). A true TPP II was, however, detected in platelets. Two purified TPP II enzymes were investigated but they did not release GPE from IGF-I under a variety of conditions. Consequently, TPP II seemed unlikely to participate in the formation of des(1-3)IGF-I. In contrast, an acidic tripeptidyl peptidase activity (TPP I) was detected in brain and colostrum, the former with a pH optimum of 4.5 and the latter 3.8. It seems likely that such an enzyme would participate in the formation of des( 1-3 )IGF-I in these tissues in vitro, ie. that des(1-3)IGF-I may have been produced as an artifact in the isolation of IGF-I from brain and colostrum in acidic conditions. This contrasts with suggestions of an in vivo role for des(1-3)IGF-I, as reported by others. The activity of a dipeptidyl peptidase N (DPP N) from urine, which should release the dipeptide GP from IGF-I, was assessed under a variety of conditions and with a variety of additives and potential enzyme stimulants, but there was no release of GP. The DPP N also exhibited a transferase activity with synthetic substrates in the presence of dipeptides, at lower concentrations than previously reported for other acceptors or other proteolytic enzymes. In addition, a low concentration of a product,possibly the tetrapeptide Gly-Pro-Gly-Leu, was detected with the action of the enzyme on IGF-I in the presence of the dipeptide Gly-Leu. As part of attempts to detect tissue production of des(1-3)IGF-I, a monoclonal antibody (MAb ), directed towards the GPE- end ofiGF-I was produced by immunisation with a 10-mer covalently attached to a carrier protein. By the use of indirect ELISA and inhibitor studies, the MAb was shown to selectively recognise peptides with anNterminal GPE- sequence, and applied to the indirect detection of des(1-3)IGF-I. The concentration of GPE in brain, measured by mass spectrometry ( MS), was low, and the concentration of total IGF-I (measured by ELISA with a commercial polyclonal antibody [P Ab]) was 40 times higher at 50 nmol/kg. This also, was not consistent with the action of a tripeptidyl peptidase in brain that converted all IGF-I to des(1-3)IGF-I plus GPE. Contrasting ELISA results, using the MAb prepared in this study, suggest an even higher concentration of intact IGF-I of 150 nmollkg. This would argue against the presence of any des( 1-3 )IGF-I in brain, but in turn, this indicates either the presence of other substances containing a GPE amino-terminus or other cross reacting epitope. Although the results of the specificity studies reported in Chapter 5 would make this latter possibility seem unlikely, it cannot be completely excluded. No GP was detected in brain by MS. No GPE was detected in colostrum by capillary electrophoresis (CE) but the interference from extraneous substances reduced the detectability of GPE by CE and this approach would require further, prior, purification and concentration steps. A molecule, with a migration time equal to that of the peptide GP, was detected in colostrum by CE, but the concentration (~ 10 11mo/L) was much higher than the IGF-I concentration measured by radio-immunoassay using a PAb (80 nmol/L) or using a Mab (300-400 nmolL). A DPP IV enzyme was detected in colostrum and this could account for the GP, derived from substrates other than IGF-1. Based on the differential results of the two antibody assays, there was no indication of the presence of des(1-3)IGF-I in brain or colostrum. In the absence of any enzyme activity directed towards the amino terminus of IGF-I and the absence any potential products, IGF-I, therefore, does not appear to "mature" via des(1-3)IGF-I in the brain, nor in the neutral colostrum. In spite of these results which indicate the absence of an enzymic attack on IGF-I and the absence of the expected products in tissues, the possibility that the conversion of IGF-I may occur in neutral conditions in limited amounts, cannot be ruled out. It remains possible that in the extracellular environment of the membrane, a complex interaction of IGF-I, binding protein, aminopeptidase(s) and receptor, produces des(1- 3)IGF-I as a transient product which is bound to the receptor and internalised.

Rationalizing the SER spectra of bacteria

The SER spectra of riboflavin and FAD are identical and are resonance enhanced at 514 or 532 nm. Signals from FAD/ riboflavin dominated SER spectra whenever these compounds were present with proteins or bacteria. SER spectra of very different bacteria such as Pseudomonas. aeruginosa, Bacillu. subtilis and Geobacillus. stearothermophilus were dominated by signals from FAD, even when these bacteria were added to a preformed colloid. The SERS signal of FAD is greatly reduced at 785 nm, and SER spectra of bacteria excited at 785 nm are quite different than those collected at 514 or 532 nm. This supports the assignment of the peaks in the 514 nm SER spectra of bacteria to FAD rather to amino acids or N-acetylglucosamine. The SER spectra of certain mixes of adenine and FAD showed similar changes to those of bacteria when the excitation was changed from 514/532 nm to 785 nm. The ratio of colloid: bacteria was of critical important for obtaining good SER spectra, and the addition of sodium sulfate was also beneficial. Removal of EPS from bacteria before analysis facilitated interaction with the silver surface, and may be a useful step to include in identification protocols.

The feasibility of vibration analysis as a technique to detect osseointegration of transfemoral implants

One of the main causes of above knee or transfemoral amputation (TFA) in the developed world is trauma to the limb. The number of people undergoing TFA due to limb trauma, particularly due to war injuries, has been increasing. Typically the trauma amputee population, including war-related amputees, are otherwise healthy, active and desire to return to employment and their usual lifestyle. Consequently there is a growing need to restore long-term mobility and limb function to this population. Traditionally transfemoral amputees are provided with an artificial or prosthetic leg that consists of a fabricated socket, knee joint mechanism and a prosthetic foot. Amputees have reported several problems related to the socket of their prosthetic limb. These include pain in the residual limb, poor socket fit, discomfort and poor mobility. Removing the socket from the prosthetic limb could eliminate or reduce these problems. A solution to this is the direct attachment of the prosthesis to the residual bone (femur) inside the residual limb. This technique has been used on a small population of transfemoral amputees since 1990. A threaded titanium implant is screwed in to the shaft of the femur and a second component connects between the implant and the prosthesis. A period of time is required to allow the implant to become fully attached to the bone, called osseointegration (OI), and be able to withstand applied load; then the prosthesis can be attached. The advantages of transfemoral osseointegration (TFOI) over conventional prosthetic sockets include better hip mobility, sitting comfort and prosthetic retention and fewer skin problems on the residual limb. However, due to the length of time required for OI to progress and to complete the rehabilitation exercises, it can take up to twelve months after implant insertion for an amputee to be able to load bear and to walk unaided. The long rehabilitation time is a significant disadvantage of TFOI and may be impeding the wider adoption of the technique. There is a need for a non-invasive method of assessing the degree of osseointegration between the bone and the implant. If such a method was capable of determining the progression of TFOI and assessing when the implant was able to withstand physiological load it could reduce the overall rehabilitation time. Vibration analysis has been suggested as a potential technique: it is a non destructive method of assessing the dynamic properties of a structure. Changes in the physical properties of a structure can be identified from changes in its dynamic properties. Consequently vibration analysis, both experimental and computational, has been used to assess bone fracture healing, prosthetic hip loosening and dental implant OI with varying degrees of success. More recently experimental vibration analysis has been used in TFOI. However further work is needed to assess the potential of the technique and fully characterise the femur-implant system. The overall aim of this study was to develop physical and computational models of the TFOI femur-implant system and use these models to investigate the feasibility of vibration analysis to detect the process of OI. Femur-implant physical models were developed and manufactured using synthetic materials to represent four key stages of OI development (identified from a physiological model), simulated using different interface conditions between the implant and femur. Experimental vibration analysis (modal analysis) was then conducted using the physical models. The femur-implant models, representing stage one to stage four of OI development, were excited and the modal parameters obtained over the range 0-5kHz. The results indicated the technique had limited capability in distinguishing between different interface conditions. The fundamental bending mode did not alter with interfacial changes. However higher modes were able to track chronological changes in interface condition by the change in natural frequency, although no one modal parameter could uniquely distinguish between each interface condition. The importance of the model boundary condition (how the model is constrained) was the key finding; variations in the boundary condition altered the modal parameters obtained. Therefore the boundary conditions need to be held constant between tests in order for the detected modal parameter changes to be attributed to interface condition changes. A three dimensional Finite Element (FE) model of the femur-implant model was then developed and used to explore the sensitivity of the modal parameters to more subtle interfacial and boundary condition changes. The FE model was created using the synthetic femur geometry and an approximation of the implant geometry. The natural frequencies of the FE model were found to match the experimental frequencies within 20% and the FE and experimental mode shapes were similar. Therefore the FE model was shown to successfully capture the dynamic response of the physical system. As was found with the experimental modal analysis, the fundamental bending mode of the FE model did not alter due to changes in interface elastic modulus. Axial and torsional modes were identified by the FE model that were not detected experimentally; the torsional mode exhibited the largest frequency change due to interfacial changes (103% between the lower and upper limits of the interface modulus range). Therefore the FE model provided additional information on the dynamic response of the system and was complementary to the experimental model. The small changes in natural frequency over a large range of interface region elastic moduli indicated the method may only be able to distinguish between early and late OI progression. The boundary conditions applied to the FE model influenced the modal parameters to a far greater extent than the interface condition variations. Therefore the FE model, as well as the experimental modal analysis, indicated that the boundary conditions need to be held constant between tests in order for the detected changes in modal parameters to be attributed to interface condition changes alone. The results of this study suggest that in a clinical setting it is unlikely that the in vivo boundary conditions of the amputated femur could be adequately controlled or replicated over time and consequently it is unlikely that any longitudinal change in frequency detected by the modal analysis technique could be attributed exclusively to changes at the femur-implant interface. Therefore further development of the modal analysis technique would require significant consideration of the clinical boundary conditions and investigation of modes other than the bending modes.

Alkaline hydrothermal treatment of titanate nanostructures

Since its initial proposal in 1998, alkaline hydrothermal processing has rapidly become an established technology for the production of titanate nanostructures. This simple, highly reproducible process has gained a strong research following since its conception. However, complete understanding and elucidation of nanostructure phase and formation have not yet been achieved. Without fully understanding phase, formation, and other important competing effects of the synthesis parameters on the final structure, the maximum potential of these nanostructures cannot be obtained. Therefore this study examined the influence of synthesis parameters on the formation of titanate nanostructures produced by alkaline hydrothermal treatment. The parameters included alkaline concentration, hydrothermal temperature, the precursor material‘s crystallite size and also the phase of the titanium dioxide precursor (TiO2, or titania). The nanostructure‘s phase and morphology was analysed using X-ray diffraction (XRD), Raman spectroscopy and transmission electron microscopy. X-ray photoelectron spectroscopy (XPS), dynamic light scattering (non-invasive backscattering), nitrogen sorption, and Rietveld analysis were used to determine phase, for particle sizing, surface area determinations, and establishing phase concentrations, respectively. This project rigorously examined the effect of alkaline concentration and hydrothermal temperature on three commercially sourced and two self-prepared TiO2 powders. These precursors consisted of both pure- or mixed-phase anatase and rutile polymorphs, and were selected to cover a range of phase concentrations and crystallite sizes. Typically, these precursors were treated with 5–10 M sodium hydroxide (NaOH) solutions at temperatures between 100–220 °C. Both nanotube and nanoribbon morphologies could be produced depending on the combination of these hydrothermal conditions. Both titania and titanate phases are comprised of TiO6 units which are assembled in different combinations. The arrangement of these atoms affects the binding energy between the Ti–O bonds. Raman spectroscopy and XPS were therefore employed in a preliminary study of phase determination for these materials. The change in binding energy from a titania to a titanate binding energy was investigated in this study, and the transformation of titania precursor into nanotubes and titanate nanoribbons was directly observed by these methods. Evaluation of the Raman and XPS results indicated a strengthening in the binding energies of both the Ti (2p3/2) and O (1s) bands which correlated to an increase in strength and decrease in resolution of the characteristic nanotube doublet observed between 320 and 220 cm.1 in the Raman spectra of these products. The effect of phase and crystallite size on nanotube formation was examined over a series of temperatures (100.200 �‹C in 20 �‹C increments) at a set alkaline concentration (7.5 M NaOH). These parameters were investigated by employing both pure- and mixed- phase precursors of anatase and rutile. This study indicated that both the crystallite size and phase affect nanotube formation, with rutile requiring a greater driving force (essentially �\harsher. hydrothermal conditions) than anatase to form nanotubes, where larger crystallites forms of the precursor also appeared to impede nanotube formation slightly. These parameters were further examined in later studies. The influence of alkaline concentration and hydrothermal temperature were systematically examined for the transformation of Degussa P25 into nanotubes and nanoribbons, and exact conditions for nanostructure synthesis were determined. Correlation of these data sets resulted in the construction of a morphological phase diagram, which is an effective reference for nanostructure formation. This morphological phase diagram effectively provides a .recipe book�e for the formation of titanate nanostructures. Morphological phase diagrams were also constructed for larger, near phase-pure anatase and rutile precursors, to further investigate the influence of hydrothermal reaction parameters on the formation of titanate nanotubes and nanoribbons. The effects of alkaline concentration, hydrothermal temperature, crystallite phase and size are observed when the three morphological phase diagrams are compared. Through the analysis of these results it was determined that alkaline concentration and hydrothermal temperature affect nanotube and nanoribbon formation independently through a complex relationship, where nanotubes are primarily affected by temperature, whilst nanoribbons are strongly influenced by alkaline concentration. Crystallite size and phase also affected the nanostructure formation. Smaller precursor crystallites formed nanostructures at reduced hydrothermal temperature, and rutile displayed a slower rate of precursor consumption compared to anatase, with incomplete conversion observed for most hydrothermal conditions. The incomplete conversion of rutile into nanotubes was examined in detail in the final study. This study selectively examined the kinetics of precursor dissolution in order to understand why rutile incompletely converted. This was achieved by selecting a single hydrothermal condition (9 M NaOH, 160 °C) where nanotubes are known to form from both anatase and rutile, where the synthesis was quenched after 2, 4, 8, 16 and 32 hours. The influence of precursor phase on nanostructure formation was explicitly determined to be due to different dissolution kinetics; where anatase exhibited zero-order dissolution and rutile second-order. This difference in kinetic order cannot be simply explained by the variation in crystallite size, as the inherent surface areas of the two precursors were determined to have first-order relationships with time. Therefore, the crystallite size (and inherent surface area) does not affect the overall kinetic order of dissolution; rather, it determines the rate of reaction. Finally, nanostructure formation was found to be controlled by the availability of dissolved titanium (Ti4+) species in solution, which is mediated by the dissolution kinetics of the precursor.

Support for gold open access publishing strategies at QUT

INTRODUCTION: Since the introduction of its QUT ePrints institutional repository of published research outputs, together with the world’s first mandate for author contributions to an institutional repository, Queensland University of Technology (QUT) has been a leader in support of green road open access. With QUT ePrints providing our mechanism for supporting the green road to open access, QUT has since then also continued to expand its secondary open access strategy supporting gold road open access, which is also designed to assist QUT researchers to maximise the accessibility and so impact of their research. ---------- METHODS: QUT Library has adopted the position of selectively supporting true gold road open access publishing by using the Library Resource Allocation budget to pay the author publication fees for QUT authors wishing to publish in the open access journals of a range of publishers including BioMed Central, Public Library of Science and Hindawi. QUT Library has been careful to support only true open access publishers and not those open access publishers with hybrid models which “double dip” by charging authors publication fees and libraries subscription fees for the same journal content. QUT Library has maintained a watch on the growing number of open access journals available from gold road open access publishers and their increased rate of success as measured by publication impact. ---------- RESULTS: This paper reports on the successes and challenges of QUT’s efforts to support true gold road open access publishers and promote these publishing strategy options to researchers at QUT. The number and spread of QUT papers submitted and published in the journals of each publisher is provided. Citation counts for papers and authors are also presented and analysed, with the intention of identifying the benefits to accessibility and research impact for early career and established researchers.---------- CONCLUSIONS: QUT Library is eager to continue and further develop support for this publishing strategy, and makes a number of recommendations to other research institutions, on how they can best achieve success with this strategy.

Is a mango just a mango? Testing within-fruit oviposition site choice and larval performance of a highly polyphagous fruit fly

For fruit flies, fully ripe fruit is preferred for adult oviposition and is superior for offspring performance over unripe or ripening fruit. Because not all parts of a single fruit ripen simultaneously, the opportunity exists for adult fruit flies to selectively choose riper parts of a fruit for oviposition and such selection, if it occurs, could positively influence offspring performance. Such fine scale host variation is rarely considered in fruit fly ecology, however, especially for polyphagous species which are, by definition, considered to be generalist host users. Here we study the adult oviposition preference/larval performance relationship of the Oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), a highly polyphagous pest species, at the “within-fruit” level to see if such a host use pattern occurs. We recorded the number of oviposition attempts that female flies made into three fruit portions (top, middle and bottom), and larval behavior and development within different fruit portions for ripening (color change) and fully-ripe mango, Mangifera indica L. (Anacardiaceae). Results indicate that female B. dorsalis do not oviposit uniformly across a mango fruit, but lay most often in the top (i.e., stalk end) of fruit and least in the bottom portion, regardless of ripening stage. There was no evidence of larval feeding site preference or performance (development time, pupal weight, percent pupation) being influenced by fruit portion, within or across the fruit ripening stages. There was, however, a very significant effect on adult emergence rate from pupae, with adult emergence rate from pupae from the bottom of ripening mango being approximately only 50% of the adult emergence rate from the top of ripening fruit, or from both the top and bottom of fully-ripe fruit. Differences in mechanical (firmness) and chemical (total soluble solids, titratable acidity, total non-structural carbohydrates) traits between different fruit portions were correlated with adult fruit utilisation. Our results support a positive adult preference/offspring performance relationship at within-fruit level for B. dorsalis. The fine level of host discrimination exhibited by B. dorsalis is at odds with the general perception that, as a polyphagous herbivore, the fly should show very little discrimination in its host use behavior.

Exposure of pipe insulation to high temperatures from domestic pumped storage (split) solar water heating systems in Australia and New Zealand

Pipe insulation between the collector and storage tank on pumped storage (commonly called split), solar water heaters can be subject to high temperatures, with a maximum equal to the collector stagnation temperature. The frequency of occurrence of these temperatures is dependent on many factors including climate, hot water demand, system size and efficiency. This paper outlines the findings of a computer modelling study to quantify the frequency of occurrence of pipe temperatures of 80 degrees Celsius or greater at the outlet of the collectors for these systems. This study will help insulation suppliers determine the suitability of their materials for this application. The TRNSYS program was used to model the performance of a common size of domestic split solar system, using both flat plate and evacuated tube, selective surface collectors. Each system was modelled at a representative city in each of the 6 climate zones for Australia and New Zealand, according to AS/NZS4234 - Heat Water Systems - Calculation of energy consumption, and the ORER RECs calculation method. TRNSYS was used to predict the frequency of occurrence of the temperatures that the pipe insulation would be exposed to over an average year, for hot water consumption patterns specified in AS/NZS4234, and for worst case conditions in each of the climate zones. The results show; * For selectively surfaced, flat plate collectors in the hottest location (Alice Sprints) with a medium size hot water demand according to AS/NZS2434, the annual frequency of occurrence of temperatures at and above 80 degrees Celsius was 33 hours. The frequency of temperatures at and above 140 degrees Celsius was insignificant. * For evacuated tube collectors in the hottest location (Alice Springs), the annual frequency of temperatures at and above 80 degrees Celsius was 50 hours. Temperatures at and above 140 degrees Celsius were significant and were estimated to occur for more than 21 hours per year in this climate zone. Even in Melbourne, temperatures at and above 80 degrees can occur for 12 hours per year and at and above 140 degrees for 5 hours per year. * The worst case identified was for evacuated tube collectors in Alice Springs, with mostly afternoon loads in January. Under these conditions, the frequency of temperatures at and above 80 degrees Celsius was 10 hours for this month only. Temperatures at and above 140 degrees Celsius were predicted to occur for 5 hours in January.

Energy and cost savings from pipe insulation on domestic, pumped storage (split), solar water heating systems in Australia and New Zealand

Appropriate pipe insulation on domestic, pumped storage (split), solar water heating systems forms an integral part of energy conservation measures of well engineered systems. However, its importance over the life of the system is often overlooked. This study outlines the findings of computer modelling to quantify the energy and cost savings by using pipe insulation between the collector and storage tank. System sizes of 270 Litre storage tank, together with either selectively surfaced, flat plate collectors (4m2 area), or 30 evacuated tube collectors, were used. Insulation thicknesses of 13mm and 15mm, pipe runs both ways of 10, 15 and 20 metres and both electric and gas boosting of systems were all considered. The TRNSYS program was used to model the system performance at a representative city in each of the 6 climate zones for Australia and New Zealand, according to AS/NZS4234 – Heat Water Systems – Calculation of energy consumption and the ORER RECs calculation method. The results show:  Energy savings from pipe insulation are very significant, even in mild climates such as Rockhampton. Across all climates zones, savings ranged from 0.16 to 3.5GJ per system per year, or about 2 to 23 percent of the annual load.  There is very little advantage in increasing the insulation thickness from 13 to 15mm. For electricity at 19c/kWh and gas at 2 c/MJ, cost savings of between $27 and $100 per year are achieved across the climate zones. Both energy and cost savings would increase in colder climates with increased system size, solar contribution and water temperatures.  The pipe insulation substantially improves the solar contribution (or fraction) and Renewable Energy Certificates (RECs), as well as giving small savings in circulating pump running costs in milder climates. Solar contribution increased by up to 23 percent points and RECs by over 7 in some cases.  The study highlights the need to install and maintain the integrity of appropriate pipe insulation on solar water heaters over their life time in Australia and New Zealand.

Green, Greener, BPM?

I am sure you’ve heard it too: Green is the new Black. While this was true back in the days when Henry Ford introduced process standardization with his assembly line for the Ford Model T (over 15 million of these were sold!), Green is also the color of choice for many business organizations, private and public. I am not talking about the actual color of their business shirts or their logo 2.0.; I am referring to the eco-aware movement that has pushed sustainability into the top ten list of business buzz-words. What used to be a boutique market for tourism and political activists has become the biggest business revolution since the e-commerce boom. Public and private organizations alike push towards “sustainable” solutions and practices. That push is partly triggered by the immense reputational gains associated with branding your organization as “green”, and partly by emerging societal, legal and constitutional regulations that force organizations to become more ecologically aware and sustainable. But the boom goes beyond organizational reality. Even in academia, sustainability has become a research “fashion wave” (see [1] if you are interested in research fashion waves) similar to the hype around Neuroscience that our colleagues in the natural sciences are witnessing these days. Mind you, I’m a fan. A big fan in fact. As academics, we are constantly searching for problem areas that are characterized by an opportunity to do rigorous research (studies that are executed to perfection) on relevant topics (studies that have applied practical value and provide impact to the community). What would be a better playground than exploring the options that Business Process Management provides for creating a sustainable, green future? I’m getting excited just writing about this! So, join me in exploring some of the current thoughts around how BPM can contribute to the sustainability fashion parade and let me introduce you to some of the works that scholars have produced recently in their attempts to identify solutions.

Near infrared optical materials from polymeric amorphous carbon synthesized by collisional plasma process

The synthesis of polymerlike amorphous carbon(a-C:H) thin-films by microwave excited collisional hydrocarbon plasma process is reported. Stable and highly aromatic a-C:H were obtained containing significant inclusions of poly(p-phenylene vinylene) (PPV). PPV confers universal optoelectronic properties to the synthesized material. That is a-C:H with tailor-made refractive index are capable of becoming absorption-free in visible (red)-near infrared wavelength range. Production of large aromatic hydrocarbon including phenyl clusters and/or particles is attributed to enhanced coagulation of elemental plasma species under collisional plasma conditions. Detailed structural and morphological changes that occur in a-C:H during the plasma synthesis are also described.

Cholesterol enhances phospholipid-dependent activated protein C anticoagulant activity

The influence of cholesterol on activated protein C (APC) anticoagulant activity in plasma and on factor Va inactivation was investigated. Anticoagulant and procoagulant activities of phosphatidylcholine/phosphatidylserine (PC/PS) vesicles containing cholesterol were assessed in the presence and absence of APC using factor Xa-1-stage clotting and factor Va inactivation assays. Cholesterol at approximate physiological membrane levels (30%) in PC/PS (60%/10% w/w) vesicles prolonged the factor Xa-1-stage clotting time dose-dependently in the presence of APC but not in the absence of APC. APC-mediated cleavage of purified recombinant factor Va variants that were modified at specific APC cleavage sites (Q306/Q679-factor Va; Q506/Q679-factor Va) was studied to define the effects of cholesterol on APC cleavage at R506 and R306. When compared to control PC/PS vesicles, cholesterol in PC/PS vesicles enhanced factor Va inactivation and the rate of APC cleavage at both R506 and R306. Cholesterol also enhanced APC cleavage rates at R306 in the presence of the APC cofactor, protein S. In summary, APC anticoagulant activity in plasma and factor Va inactivation as a result of cleavages at R506 and R306 by APC is markedly enhanced by cholesterol in phospholipid vesicles. These results suggest that cholesterol in a membrane surface may selectively enhance APC activities. © 2005 International Society on Thrombosis and Haemostasis.

Porphyrin containing isoindoline nitroxides as potential fluorescence sensors of free radicals

A series of new spin-labeled porphyrin containing isoindoline nitroxide moieties were synthesized and characterized as potential free radical fluorescence sensors. Fluorescence-suppression was observed in the free-base monoradical porphyrins, whilst the free-base biradical porphyrins exhibited highly suppressed fluorescence about three times greater than the monoradical porphyrins. The observed fluorescence-suppression was attributed to enhanced intersystem crossing resulting from electronexchange between the doublet nitroxide and the excited porphyrin fluorophore. Notably, fluorescencesuppression was not as strong in the related metalated porphyrins, possibly due to insufficient spin coupling between the nitroxide and the porphyrin. Continuous wave EPR spectroscopy of the diradical porphyrins in fluid solution suggests that the nitroxyl-nitroxyl interspin distance is long enough and tumbling is fast enough not to detect dipolar coupling.

Partial agonists of the [alpha]3[beta]4[ast] neuronal nicotinic acetylcholine receptor reduce ethanol consumption and seeking in rats

Alcohol use disorders (AUDs) impact millions of individuals and there remain few effective treatment strategies. Despite evidence that neuronal nicotinic acetylcholine receptors (nAChRs) have a role in AUDs, it has not been established which subtypes of the nAChR are involved. Recent human genetic association studies have implicated the gene cluster CHRNA3-CHRNA5-CHRNB4 encoding the α3, α5, and β4 subunits of the nAChR in susceptibility to develop nicotine and alcohol dependence; however, their role in ethanol-mediated behaviors is unknown due to the lack of suitable and selective research tools. To determine the role of the α3, and β4 subunits of the nAChR in ethanol self-administration, we developed and characterized high-affinity partial agonists at α3β4 nAChRs, CP-601932, and PF-4575180. Both CP-601932 and PF-4575180 selectively decrease ethanol but not sucrose consumption and operant self-administration following long-term exposure. We show that the functional potencies of CP-601932 and PF-4575180 at α3β4 nAChRs correlate with their unbound rat brain concentrations, suggesting that the effects on ethanol self-administration are mediated via interaction with α3β4 nAChRs. Also varenicline, an approved smoking cessation aid previously shown to decrease ethanol consumption and seeking in rats and mice, reduces ethanol intake at unbound brain concentrations that allow functional interactions with α3β4 nAChRs. Furthermore, the selective α4β2(*) nAChR antagonist, DHβE, did not reduce ethanol intake. Together, these data provide further support for the human genetic association studies, implicating CHRNA3 and CHRNB4 genes in ethanol-mediated behaviors. CP-601932 has been shown to be safe in humans and may represent a potential novel treatment for AUDs.

Dopamine responsiveness is regulated by targeted sorting of D2 receptors

Abstract Aberrant dopaminergic signaling is a critical determinant in multiple psychiatric disorders, and in many disease states, dopamine receptor number is altered. Here we identify a molecular mechanism that selectively targets D2 receptors for degradation after their activation by dopamine. The degradative fate of D2 receptors is determined by an interaction with G protein coupled receptor-associated sorting protein (GASP). As a consequence of this GASP interaction, D2 responses in rat brain fail to resensitize after agonist treatment. Disruption of the D2-GASP interaction facilitates recovery of D2 responses, suggesting that modulation of the D2-GASP interaction is important for the functional down-regulation of D2 receptors.