Identification of an attractin-like pheromone in the mucus-secreting hypobranchial gland of the abalone Haliotis asinina linnaeus.

Pheromones are chemicals used to communicate between animals of the same species, and are thought to be used by most marine animals. With limited vision, abalone primarily sense their world chemically, and pheromones may play an important role in settlement, attraction, recognition, alarm, and reproduction. Despite this, there has been no detailed investigation into pheromone substances, both in their precise biochemical nature or pheromonal function. In this study, we investigated the presence of pheromonelike substances from the hypobranchial gland of the abalone Haliotis asinina using bioassays, immunohistochemistry, Western blotting, and reverse-phase high-performance liquid chromatography (RP-HPLC). The hypobranchial gland of many prosobranchial marine molluscs has been classified as a sex auxiliary gland releasing unknown substances during spawning. In our study, cephalic tentacle assays demonstrated that the cell extracts of the hypobranchial gland contain chemical cues that are sensed by conspecifics. An antibody against the sea slug “attractin” pheromone was used as a probe to localize a similar protein in the mucin-secreting cells of the epithelial lining the hypobranchial gland of both male and female abalone. The approximate molecular weight of this abalone attractin-like protein is 30 kDa in both males and females. Fractionation of hypobranchial gland extracts by C5 RP-HPLC could not selectively purify this protein, and no sex-specific differences were observed. We predict that the attractin-like protein could be one of a number of important proteins involved in maturation, aggregation, and/or spawning behavior of abalone. In future research, additional hypobranchial gland components will be tested further for these types of behavior.

The concentration of oleocanthal in olive oil waste

The aim of this study was to determine the concentration of oleocanthal in olive pomace waste and compare this to its concentration in extra-virgin olive oil (EVOO). The concentration of oleocanthal in freshly pressed EVOO and its subsequent waste was analysed at early, mid and late season harvests. Oleocanthal concentrations were quantified using high-performance liquid chromatography–mass spectrometry. In oil, oleocanthal concentration was as follows: 123.24 ± 6.48 mg kg¯¹1 in early harvest, 114.20 ± 17.42 mg kg¯¹ in mid harvest and 152.22 ± 10.54 mg kg¯¹ in late harvest. Its concentration in waste was determined to be: 128.25 ± 11.33 mg kg¯¹ in early harvest, 112.15 ± 1.51mg kg¯¹ in mid harvest and 62.35 ± 8.00 mg kg¯¹ in late harvest. Overall, olive pomace waste is a valuable source of oleocanthal.

A novel cloud-point extraction process for preconcentrating selected polycyclic aromatic hydrocarbons in aqueous solution

A novel but simple cloud-point extraction (CPE) process is developed to preconcentrate the trace of selected polycyclic aromatic hydrocarbons (PAHs) with the use of the readily biodegradable nonionic surfactant Tergitol 15-S-7 as extractant. The concentrations of PAHs, mixtures of naphthalene and phenanthrene as well as pyrene, in the spiked samples were determined with the new CPE process at ambient temperature (23 °C) followed by high performance liquid chromatography (HPLC) with fluorescence detection. More than 80% of phenanthrene and pyrene, respectively, and 96% of naphthalene initially present in the aqueous solutions with concentrations near or below their aqueous solubilities were recovered using this new CPE process. Importantly Tergitol 15-S-7 does not give any fluorometric signal to interfere with fluorescence detection of PAHs in the UV range. No special washing step is, thus, required to remove surfactant before HPLC analyses. Different experimental conditions were studied. The optimum conditions for the preconcentration and determi nation of these selected PAHs at ambient temperature have been established as the following:  (1) 3 wt % surfactant; (2) addition of 0.5 M Na2SO4; (3) 10 min for equilibration time; and (4) 3000 rpm for centrifugal speed with duration of 10 min.

Detection of brassinosteroids in pollen of Lolium perenne L. by immunocytochemistry

Bioactive brassinosteroids have been localized in developing and mature pollen of anhydrously fixed rye-grass (Lolium perenne) by immunocytochemistry using polyclonal antibodies to castasterone generated in rabbits. Tricellular pollen fixed by freeze-substitution was also labelled in the starch granules. Study of the developmental sequence of the pollen through the microsporocyte, microspore, bicellular and tricellular stages showed that the brassinosteroids were increasingly sequestered in starch granules as the amyloplasts matured, supporting the view that these are storage organelles for these potent plant growth promoters. In bicellular pollen, heavy labelling was seen in the zone within 0.5 μm of the starch granule, where stromal tissue remains. Thus, the stroma may be the site of synthesis of these compounds. During aqueous fixation, the brassinosteroids leached from the starch granules of tricellular pollen, indicating that they would be quickly available after imbibition to influence the physiology of germinating pollen. The results from high-performance liquid chromatography of dansylaminophenylboronates from partially purified extracts of freshly dehisced tricellular pollen of rye-grass showed 25-methylcastasterone may be a minor component, together with two unknown peaks. No specific binding of brassinolide to any soluble proteins extracted from tricellular rye-grass pollen was observed using the antibodies in gel electrophoresis or enzyme-linked immunosorbent assays.

Variation in the gene coding for the M5 muscarinic receptor (CHRM5) influences cigarette dose but is not associated with dependence to drugs of addiction : evidence from a prospective population based cohort study of young adults

Background: The mesolimbic structures of the brain are important in the anticipation and perception of reward. Moreover, many drugs of addiction elicit their response in these structures. The M5 muscarinic receptor (M5R) is expressed in dopamine-containing neurones of the substantia nigra pars compacta and ventral tegmental area, and regulates the release of mesolimbic dopamine. Mice lacking M5R show a substantial reduction in both reward and withdrawal responses to morphine and cocaine. The CHRM5, the gene that codes for the M5R, is a strong biological candidate for a role in human addiction. We screened the coding and core promoter sequences of CHRM5 using denaturing high performance liquid chromatography to identify common polymorphisms. Additional polymorphisms within the coding and core promoter regions that were identified through dbSNP were validated in the test population. We investigated whether these polymorphisms influence substance dependence and dose in a cohort of 1947 young Australians.

Results: Analysis was performed on 815 participants of European ancestry who were interviewed at wave 8 of the cohort study and provided DNA. We observed a 26.8% increase in cigarette consumption in carriers of the rs7162140 T-allele, equating to 20.1 cigarettes per week (p=0.01). Carriers of the rs7162140 T-allele were also found to have nearly a 3-fold increased risk of developing cannabis dependence (OR=2.9 (95%CI 1.1-7.4); p=0.03).

Conclusion: Our data suggest that variation within the CHRM5 locus may play an important role in tobacco and cannabis but not alcohol addiction in European ancestry populations. This is the first study to show an association between CHRM5 and substance use in humans. These data support the further investigation of this gene as a risk factor in substance use and dependence.

Determination of antioxidants using chemiluminescence detection

Antioxidants are produced within the body or obtained from dietary sources. Their main role is to counter the detrimental effects of free radicals, but they are also essential for numerous metabolic processes. This thesis describes novel detection methods for the determination of antioxidants in plant based materials and physiological fluids.

Selectivity in separation using π electron-rich stationary phases for the comprehensive two-dimensional analysis of cafe espresso

The multidimensional high-performance liquid chromatography separations of the complex sample matrix found in café espresso coffee were completed on the propyl phenyl and butyl phenyl columns that contain 3 and 4 carbon atoms in the spacer chain, respectively. Phenyl type stationary phases are able to undergo unique π–π interactions with aromatic compounds. Previous works have found that there are differences in retention characteristics between these chain lengths and this was explored further here. It was found that when analysing the separations by quadrants, using a geometric approach to factor analysis and by measuring the normalised mean radius, subtle differences in the separations were observed and the butyl phenyl phase was more selective for the high to medium polarity species. However, there was very little difference in separation behaviour for the hydrophobic components within the coffee sample. Overall, the analysis of the entire separation showed very little difference.

1H-NMR and mass spectrometric characterization of the metabolic response of juvenile Atlantic salmon (Salmo salar) to long-term handling stress

Stressors of various kinds constantly affect fish both in the wild and in culture, examples being acute water temperature and quality changes, predation, handling, and confinement. Known physiological responses of fish to stress such as increases in plasma cortisol and glucose levels, are considered to be adaptive, allowing the animal to cope in the short term. Prolonged exposure to stressors however, has the potential to affect growth, immune function, and survival. Nonetheless, little is known about the mechanisms underlying the long-term stress response. We have investigated the metabolic response of juvenile Atlantic salmon (Salmo salar) to long-term handling stress by analyzing fish plasma via ¹H nuclear magnetic resonance spectroscopy and ultra high performance liquid chromatography–mass spectrometry (UPLC–MS), and comparing results with controls. Analysis of NMR data indicated a difference in the metabolic profiles of control and stressed fish after 1 week of stress with a maximum difference observed after 2 weeks. These differences were associated with stress-induced increases in phosphatidyl choline, lactate, carbohydrates, alanine, valine and trimethylamine-N-oxide, and decreases in low density lipoprotein, very low density lipoprotein, and lipid. UPLC-MS data showed differences at week 2, associated with another set of compounds, tentatively identified on the basis of their mass/charge. Overall the results provided a multi-faceted view of the response of fish to long-term handling stress, indicating that the metabolic disparity between the control and stress groups increased to week 2, but declined by weeks 3 and 4, and revealed several new molecular indicators of long-term stress.

π-Selective stationary phases: (III) Influence of the propyl phenyl ligand density on the aromatic and methylene selectivity of aromatic compounds in reversed phase liquid chromatography

The retention characteristics of phenyl type stationary phases for reversed phase high performance liquid chromatography are still largely unknown. This paper explores the retention process of these types of stationary phases by examining the retention behaviour of linear PAHs and n-alkylbenzenes on a series of propyl phenyl stationary phases that have changes in their ligand density (1.23, 1.31, 1.97, 2.50 μmol m⁻²). The aromatic and methylene selectivities increased with increasing ligand density until a point where a plateau was observed, overall the propyl phenyl phases had a higher degree of aromatic selectivity than methylene selectivity indicating that these columns are suitable for separations involving aromatic compounds. Also, retention characteristics relating to the size of the solute molecule were observed to be influenced by the ligand density. It is likely that the changing retention characteristics are caused by the different topologies of the stationary phases at different ligand densities. At high ligand densities, the partition coefficient became constant.

The determination of bioactives using multidimensional separation and detection systems

This project was focused on the development of platform technology for the separation and detection of complex samples. The samples investigated included roasted coffee beans, opiate alkaloids and amino acids.

Chemiluminescence detection of piperazine designer drugs and related compounds using tris(2,2?-bipyridine)ruthenium(III)

 An exploration of the chemiluminescence from reactions of a large number of benzyl and phenylpiperazine analytes with tris(2,2’-bipyridyl)ruthenium(III) was carried out providing information towards the emission intensity of this chemiluminescent reagent and the structure of analytes it interacts with.

Synthesis and biological evaluation of novel folic acid receptor-targeted, β-cyclodextrin-based drug complexes for cancer treatment

Drug targeting is an active area of research and nano-scaled drug delivery systems hold tremendous potential for the treatment of neoplasms. In this study, a novel cyclodextrin (CD)-based nanoparticle drug delivery system has been assembled and characterized for the therapy of folate receptor-positive [FR(+)] cancer. Water-soluble folic acid (FA)-conjugated CD carriers (FACDs) were successfully synthesized and their structures were confirmed by 1D/2D nuclear magnetic resonance (NMR), matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS), high performance liquid chromatography (HPLC), Fourier transform infrared spectroscopy (FTIR), and circular dichroism. Drug complexes of adamatane (Ada) and cytotoxic doxorubicin (Dox) with FACD were readily obtained by mixed solvent precipitation. The average size of FACD-Ada-Dox was 1.5-2.5 nm. The host-guest association constant Ka was 1,639 M-1 as determined by induced circular dichroism and the hydrophilicity of the FACDs was greatly enhanced compared to unmodified CD. Cellular uptake and FR binding competitive experiments demonstrated an efficient and preferentially targeted delivery of Dox into FR-positive tumor cells and a sustained drug release profile was seen in vitro. The delivery of Dox into FR(+) cancer cells via endocytosis was observed by confocal microscopy and drug uptake of the targeted nanoparticles was 8-fold greater than that of non-targeted drug complexes. Our docking results suggest that FA, FACD and FACD-Ada-Dox could bind human hedgehog interacting protein that contains a FR domain. Mouse cardiomyocytes as well as fibroblast treated with FACD-Ada-Dox had significantly lower levels of reactive oxygen species, with increased content of glutathione and glutathione peroxidase activity, indicating a reduced potential for Dox-induced cardiotoxicity. These results indicate that the targeted drug complex possesses high drug association and sustained drug release properties with good biocompatibility and physiological stability. The novel FA-conjugated β-CD based drug complex might be promising as an anti-tumor treatment for FR(+) cancer.

Determination of neurotransmitters and their metabolites using one- and two-dimensional liquid chromatography with acidic potassium permanganate chemiluminescence detection

High-performance liquid chromatography with chemiluminescence detection based on the reaction with acidic potassium permanganate and formaldehyde was explored for the determination of neurotransmitters and their metabolites. The neurotransmitters norepinephrine and dopamine were quantified in the left and right hemispheres of rat hippocampus, nucleus accumbens and prefrontal cortex, and the metabolites vanillylmandelic acid, 3,4-dihydrophenylacetic acid, 5-hydroxyindole-3-acetic acid and homovanillic acid were identified in human urine. Under optimised chemiluminescence reagent conditions, the limits of detection for these analytes ranged from 2.5 × 10−8 to 2.5 × 10−7 M. For the determination of neurotransmitter metabolites in urine, a two-dimensional high-performance liquid chromatography (2D-HPLC) separation operated in heart-cutting mode was developed to overcome the peak capacity limitations of the one-dimensional separation. This approach provided the greater separation power of 2D-HPLC with analysis times comparable to conventional one-dimensional separations.