Identification of bone morphogenetic proteins 2 and 4 in commercial demineralized freeze-dried bone allograft preparations : pilot study

BACKGROUND: Demineralized freeze-dried bone allografts (DFDBAs) have been proposed as a useful adjunct in periodontal therapy to induce periodontal regeneration through the induction of new bone formation. The presence of bone morphogenetic proteins (BMPs) within the demineralized matrix has been proposed as a possible mechanism through which DFDBA may exert its biologic effect. However, in recent years, the predictability of results using DFDBA has been variable and has led to its use being questioned. One reason for the variability in tissue response may be attributed to differences in the processing of DFDBA, which may lead to loss of activity of any bioactive substances within the DFDBA matrix. Therefore, the purpose of this investigation was to determine whether there are detectable levels of bone morphogenetic proteins in commercial DFDBA preparations. METHODS: A single preparation of DFDBA was obtained from three commercial sources. Each preparation was studied in triplicate. Proteins within the DFDBA samples were first extracted with 4M guanidinium HCI for seven days at 40 degrees celsius and the residue was further extracted with 4M guanidinium HCL/EDTA for seven days at 40 degrees celsius. Two anti-human BMP-2 and -4 antibodies were used for the detection of the presence of BMP's in the extracts. RESULTS: Neither BMP-2 nor BMP-4 was detected in any of the extracts. When recombinant human BMP-2 and -4 were added throughout the extraction process of DFDBA extraction, not only were intact proteins detected but smaller molecular weight fragments were also noted in the extract. CONCLUSIONS: These results indicate that all of the DFDBA samples tested had no detectable amounts of BMP-2 and -4. In addition, an unknown substance present in the DFDBA may be responsible for degradation of whatever BMPs might be present.

An investigation into the use of human papillomavirus type 16 virus-like particles as a delivery vector system for foreign proteins: N- and C-terminal fusion of GFP to the L1 and L2 capsid proteins

Development of vaccine strategies against human papillomavirus (HPV), which causes cervical cancer, is a priority. We investigated the use of virus-like particles (VLPs) of the most prevalent type, HPV-16, as carriers of foreign proteins. Green fluorescent protein (GFP) was fused to the N or C terminus of both L1 and L2, with L2 chimeras being co-expressed with native L1. Purified chimaeric VLPs were comparable in size (∼55 nm) to native HPV VLPs. Conformation-specific monoclonal antibodies (Mabs) bound to the VLPs, thereby indicating that they possibly retain their antigenicity. In addition, all of the VLPs encapsidated DNA in the range of 6-8 kb. © 2007 Springer-Verlag.

Ghrelin receptor (GHS-R1A) antagonism suppresses both operant alcohol self-administration and high alcohol consumption in rats

The mechanisms involved in alcohol use disorders are complex. It has been shown that ghrelin is an important signal for the control of body weight homeostasis, preferably by interacting with hypothalamic circuits, as well as for drug reward by activating the mesolimbic dopamine system. The ghrelin receptor (GHS-R1A) has been shown to be required for alcohol-induced reward. Additionally, ghrelin increases and GHR-R1A antagonists reduce moderate alcohol consumption in mice, and a single nucleotide polymorphism in the GHS-R1A gene has been associated with high alcohol consumption in humans. However, the role of central ghrelin signaling in high alcohol consumption is not known. Therefore, the role of GHS-R1A in operant self-administration of alcohol in rats as well as for high alcohol consumption in Long-Evans rats and in alcohol preferring [Alko alcohol (AA)] rats was studied here. In the present study, the GHS-R1A antagonist, JMV2959, was found to reduce the operant self-administration of alcohol in rats and to decrease high alcohol intake in Long-Evans rats as well as in AA rats. These results suggest that the ghrelin receptor signaling system, specifically GHS-R1A, is required for operant self-administration of alcohol and for high alcohol intake in rats. Therefore, the GHS-R1A may be a therapeutic target for treatment of addictive behaviors, such as alcohol dependence.

Identification of optimal epitopes for Plasmodium falciparum rapid diagnostic tests that target histidine-rich proteins 2 and 3

Rapid diagnostic tests (RDTs) represent important tools to diagnose malaria infection. To improve understanding of the variable performance of RDTs that detect the major target in Plasmodium falciparum, namely, histidine-rich protein 2 (HRP2), and to inform the design of better tests, we undertook detailed mapping of the epitopes recognized by eight HRP-specific monoclonal antibodies (MAbs). To investigate the geographic skewing of this polymorphic protein, we analyzed the distribution of these epitopes in parasites from geographically diverse areas. To identify an ideal amino acid motif for a MAb to target in HRP2 and in the related protein HRP3, we used a purpose-designed script to perform bioinformatic analysis of 448 distinct gene sequences from pfhrp2 and from 99 sequences from the closely related gene pfhrp3. The frequency and distribution of these motifs were also compared to the MAb epitopes. Heat stability testing of MAbs immobilized on nitrocellulose membranes was also performed. Results of these experiments enabled the identification of MAbs with the most desirable characteristics for inclusion in RDTs, including copy number and coverage of target epitopes, geographic skewing, heat stability, and match with the most abundant amino acid motifs identified. This study therefore informs the selection of MAbs to include in malaria RDTs as well as in the generation of improved MAbs that should improve the performance of HRP-detecting malaria RDTs.

Global sequence variation in the histidine-rich proteins 2 and 3 of Plasmodium falciparum: implications for the performance of malaria rapid diagnostic tests

Background Accurate diagnosis is essential for prompt and appropriate treatment of malaria. While rapid diagnostic tests (RDTs) offer great potential to improve malaria diagnosis, the sensitivity of RDTs has been reported to be highly variable. One possible factor contributing to variable test performance is the diversity of parasite antigens. This is of particular concern for Plasmodium falciparum histidine-rich protein 2 (PfHRP2)-detecting RDTs since PfHRP2 has been reported to be highly variable in isolates of the Asia-Pacific region. Methods The pfhrp2 exon 2 fragment from 458 isolates of P. falciparum collected from 38 countries was amplified and sequenced. For a subset of 80 isolates, the exon 2 fragment of histidine-rich protein 3 (pfhrp3) was also amplified and sequenced. DNA sequence and statistical analysis of the variation observed in these genes was conducted. The potential impact of the pfhrp2 variation on RDT detection rates was examined by analysing the relationship between sequence characteristics of this gene and the results of the WHO product testing of malaria RDTs: Round 1 (2008), for 34 PfHRP2-detecting RDTs. Results Sequence analysis revealed extensive variations in the number and arrangement of various repeats encoded by the genes in parasite populations world-wide. However, no statistically robust correlation between gene structure and RDT detection rate for P. falciparum parasites at 200 parasites per microlitre was identified. Conclusions The results suggest that despite extreme sequence variation, diversity of PfHRP2 does not appear to be a major cause of RDT sensitivity variation.

Comparison of multiple and novel measures of dietary glycemic carbohydrate with insulin resistant status in older women

BACKGROUND:Previous epidemiological investigations of associations between dietary glycemic intake and insulin resistance have used average daily measures of glycemic index (GI) and glycemic load (GL). We explored multiple and novel measures of dietary glycemic intake to determine which was most predictive of an association with insulin resistance.METHODS:Usual dietary intakes were assessed by diet history interview in women aged 42-81 years participating in the Longitudinal Assessment of Ageing in Women. Daily measures of dietary glycemic intake (n = 329) were carbohydrate, GI, GL, and GL per megacalorie (GL/Mcal), while meal based measures (n = 200) were breakfast, lunch and dinner GL; and a new measure, GL peak score, to represent meal peaks. Insulin resistant status was defined as a homeostasis model assessment (HOMA) value of >3.99; HOMA as a continuous variable was also investigated.RESULTS:GL, GL/Mcal, carbohydrate (all P < 0.01), GL peak score (P = 0.04) and lunch GL (P = 0.04) were positively and independently associated with insulin resistant status. Daily measures were more predictive than meal-based measures, with minimal difference between GL/Mcal, GL and carbohydrate. No significant associations were observed with HOMA as a continuous variable.CONCLUSION:A dietary pattern with high peaks of GL above the individual's average intake was a significant independent predictor of insulin resistance in this population, however the contribution was less than daily GL and carbohydrate variables. Accounting for energy intake slightly increased the predictive ability of GL, which is potentially important when examining disease risk in more diverse populations with wider variations in energy requirements.

Investigation of electrophoretic loading and enhanced mechanical properties of hydrogels for delivery of therapeutic proteins

Hydrogels, which are three-dimensional crosslinked hydrophilic polymers, have been used and studied widely as vehicles for drug delivery due to their good biocompatibility. Traditional methods to load therapeutic proteins into hydrogels have some disadvantages. Biological activity of drugs or proteins can be compromised during polymerization process or the process of loading protein can be really timeconsuming. Therefore, different loading methods have been investigated. Based on the theory of electrophoresis, an electrochemical gradient can be used to transport proteins into hydrogels. Therefore, an electrophoretic method was used to load protein in this study. Chemically and radiation crosslinked polyacrylamide was used to set up the model to load protein electrophoretically into hydrogels. Different methods to prepare the polymers have been studied and have shown the effect of the crosslinker (bisacrylamide) concentration on the protein loading and release behaviour. The mechanism of protein release from the hydrogels was anomalous diffusion (i.e. the process was non-Fickian). The UV-Vis spectra of proteins before and after reduction show that the bioactivities of proteins after release from hydrogel were maintained. Due to the concern of cytotoxicity of residual monomer in polyacrylamide, poly(2-hydroxyethyl- methacrylate) (pHEMA) was used as the second tested material. In order to control the pore size, a polyethylene glycol (PEG) porogen was introduced to the pHEMA. The hydrogel disintegrated after immersion in water indicating that the swelling forces exceeded the strength of the material. In order to understand the cause of the disintegration, several different conditions of crosslinker concentration and preparation method were studied. However, the disintegration of the hydrogel still occurred after immersion in water principally due to osmotic forces. A hydrogel suitable for drug delivery needs to be biocompatible and also robust. Therefore, an approach to improving the mechanical properties of the porogen-containing pHEMA hydrogel by introduction of an inter-penetrating network (IPN) into the hydrogel system has been researched. A double network was formed by the introduction of further HEMA solution into the system by both electrophoresis and slow diffusion. Raman spectroscopy was used to observe the diffusion of HEMA into the hydrogel prior to further crosslinking by ã-irradiation. The protein loading and release behaviour from the hydrogel showing enhanced mechanical property was also studied. Biocompatibility is a very important factor for the biomedical application of hydrogels. Different hydrogels have been studied on both a three-dimensional HSE model and a HSE wound model for their biocompatibilities. They did not show any detrimental effect to the keratinocyte cells. From the results reported above, these hydrogels show good biocompatibility in both models. Due to the advantage of the hydrogels such as the ability to absorb and deliver protein or drugs, they have potential to be used as topical materials for wound healing or other biomedical applications.

Rice ragged stunt oryzavirus genome segments S7 and S10 encode non-structural proteins of M(r) 68 025 (Pns7) and M(r) 32364 (Pns10) : brief report

The nucleotide sequences of genome segments S7 and S10 of a Thai-isolate of rice ragged stunt virus (RRSV) were determined. The 1938 bp S7 sequence contains a single large open reading frame (ORF) spanning nucleotides 20 to 1 843 that is predicted to encode a protein of M(r) 68 025. The 1 162 bp S10 sequence has a major ORF spanning nucleotides 142 to 1 032 that is predicted to encode a protein of M(r) 32364. This S10 ORF is preceded by a small ORF (nt 20-55) which is probably a minicistron. Coupled in vitro transcription-translation from the two major ORFs gave protein products of the expected sizes. However, no protein was visualised from S10 when the small ORF sequence was included. Proteins were expressed in Escherichia coli from the full length ORF of S7 (P7) and from a segment of the S10 ORF (P10) fused to the ORF of glutathione S-transferase (GST). Neither fusion protein was recognised by polyclonal antibodies raised against RRSV particles. Furthermore, polyclonal antibodies raised against GST-P7 fusion protein did not recognise any virion structural polypeptides. These data strongly suggest that the proteins P7 and P10 do not form part of RRSV particle. This is further supported by observed sequence homology (though very weak) of predicted.

Differential subcellular and extracellular localisations of proteins required for insulin-like growth factor- and extracellular matrix-induced signalling events in breast cancer progression

Background: Cancer metastasis is the main contributor to breast cancer fatalities as women with the metastatic disease have poorer survival outcomes than women with localised breast cancers. There is an urgent need to develop appropriate prognostic methods to stratify patients based on the propensities of their cancers to metastasise. The insulin-like growth factor (IGF)-I:IGF binding protein (IGFBP):vitronectin complexes have been shown to stimulate changes in gene expression favouring increased breast cancer cell survival and a migratory phenotype. We therefore investigated the prognostic potential of these IGF- and extracellular matrix (ECM) interaction-induced proteins in the early identification of breast cancers with a propensity to metastasise using patient-derived tissue microarrays. Methods: Semiquantitative immunohistochemistry analyses were performed to compare the extracellular and subcellular distribution of IGF- and ECM-induced signalling proteins among matched normal, primary cancer and metastatic cancer formalin-fixed paraffin-embedded breast tissue samples. Results: The IGF- and ECM-induced signalling proteins were differentially expressed between subcellular and extracellular localisations. Vitronectin and IGFBP-5 immunoreactivity was lower while β1 integrin immunoreactivity was higher in the stroma surrounding metastatic cancer tissues, as compared to normal breast and primary cancer stromal tissues. Similarly, immunoreactive stratifin was found to be increased in the stroma of primary as well as metastatic breast tissues. Immunoreactive fibronectin and β1 integrin was found to be highly expressed at the leading edge of tumours. Based on the immunoreactivity it was apparent that the cell signalling proteins AKT1 and ERK1/2 shuffled from the nucleus to the cytoplasm with tumour progression. Conclusion: This is the first in-depth, compartmentalised analysis of the distribution of IGF- and ECM-induced signalling proteins in metastatic breast cancers. This study has provided insights into the changing pattern of cellular localisation and expression of IGF- and ECM-induced signalling proteins in different stages of breast cancer. The differential distribution of these biomarkers could provide important prognostic and predictive indicators that may assist the clinical management of breast disease, namely in the early identification of cancers with a propensity to metastasise, and/or recur following adjuvant therapy.

Structural analysis of the roles of influenza A virus membrane-associated proteins in assembly and morphology

The assembly of influenza A virus at the plasma membrane of infected cells leads to release of enveloped virions that are typically round in tissue culture-adapted strains but filamentous in strains isolated from patients. The viral proteins hemagglutinin (HA), neuraminidase (NA), matrix protein 1 (M1), and M2 ion channel all contribute to virus assembly. When expressed individually or in combination in cells, they can all, under certain conditions, mediate release of membrane-enveloped particles, but their relative roles in virus assembly, release, and morphology remain unclear. To investigate these roles, we produced membrane-enveloped particles by plasmid-derived expression of combinations of HA, NA, and M proteins (M1 and M2) or by infection with influenza A virus. We monitored particle release, particle morphology, and plasma membrane morphology by using biochemical methods, electron microscopy, electron tomography, and cryo-electron tomography. Our data suggest that HA, NA, or HANA (HA plus NA) expression leads to particle release through nonspecific induction of membrane curvature. In contrast, coexpression with the M proteins clusters the glycoproteins into filamentous membrane protrusions, which can be released as particles by formation of a constricted neck at the base. HA and NA are preferentially distributed to differently curved membranes within these particles. Both the budding intermediates and the released particles are morphologically similar to those produced during infection with influenza A virus. Together, our data provide new insights into influenza virus assembly and show that the M segment together with either of the glycoproteins is the minimal requirement to assemble and release membrane-enveloped particles that are truly virus-like.

Tax morale affects tax compliance : evidence from surveys and an artefactual field experiment

Our working hypotheses is that cross-cultural differences in tax compliance behaviour have foundations in the institutions of tax administration and citizen assessment of the quality of governance. Tax compliance being a complex behavioural issue. Its investigation requires use of a variety of methods and data sources. Results from artefactual field experiments conducted in countries with substantially different political histories and records of governance quality demondtrate that observed differences in tax compliance levels persist over alternative levels of enforcement. The experimental results are shown to be robust by replicating them for the same countries using survey response measures of tax compliance.

Drugs, dental professionals and the law

Dentists have the privilege of possessing, administering and prescribing drugs, including highly addictive medications, to their patients. But because drugs are often vulnerable to being abused by all members of society, including dentists and their patients, and because drugs can be dangerous, they are tightly regulated in Canada by the federal and provincial/territorial governments. Regulatory and professional dental bodies also provide guidance for their members about how to best administer and prescribe drugs. This chapter outlines the regulation by federal and provincial/territorial governments in this area, examines the professional practice requirements set out by regulatory/professional bodies and the issue of drug abuse by dental professional and patients. It is important to note from the outset that governmental and professional regulations, policies and practices differ from province to province and territory to territory. This chapter aims to alert dentists to possible legal and professional issues surrounding the possession, administration and prescription of drugs. For detailed specific information about regulation, policies, ethical standards and professional practice standards in Canada or their province/ territory, dentists should contact their insurer or professional association.

No energy compensation at the meal following exercise indietary restrained and unrestrained women

The objective of this investigation was to compare the acute effects of exercise and diet manipulations on energy intake, between dietary restrained and unrestrained females. Comparisons of two studies using an identical 2 x 2 repeated-measures design (level of activity (rest or exercise) and lunch type (high-fat or low-fat)) including thirteen dietary unrestrained and twelve restrained females were performed. Energy expenditure during the rest session was estimated and the energy cost of exercise was measured by indirect calorimetry. Relative energy intake was calculated by subtracting the energy expenditure of the exercise session from the energy intake of the test meal. Post-meal hedonic ratings were completed after lunch. Energy intake and relative energy intake increased during high-fat conditions compared with the low-fat, independently of exercise (P < 0.001). There was a positive relationship between dietary restraint scores and energy intake or relative energy intake in the rest conditions only (r 0.54, P < 0.01). The decrease of relative energy intake between the rest and exercise conditions was higher in restrained than in unrestrained eaters (P < 0.01). These results confirm that a high-fat diet reversed the energy deficit due to exercise. There was no energy compensation in response to an acute bout of exercise during the following meal. In restrained eaters, exercise was more effective in creating an energy deficit than in unrestrained eaters. Exercise may help restrained eaters to maintain control over appetite.

Didiman: Australian agricultural extension officers in the territory of Papua and New Guinea, 1945-1975

Historically, the development philosophy for the two Territories of Papua and New Guinea (known as TPNG, formerly two territories, Papua and New Guinea) was equated with economic development, with a focus on agricultural development. To achieve the modification or complete change in indigenous farming systems the Australian Government’s Department of External Territories adopted and utilised a programme based on agricultural extension. Prior to World War II, under Australian administration, the economic development of these two territories, as in many colonies of the time, was based on the institution of the plantation. Little was initiated in agriculture development for indigenous people. This changed after World War II to a rationale based on the promotion and advancement of primary industry, but also came to include indigenous farmers. To develop agriculture within a colony it was thought that a modification to, or in some cases the complete transformation of, existing farming systems was necessary to improve the material welfare of the population. It was also seen to be a guarantee for the future national interest of the sovereign state after independence was granted. The Didiman and Didimisis became the frontline, field operatives of this theoretical model of development. This thesis examines the Didiman’s field operations, the structural organisation of agricultural administration and the application of policy in the two territories.