Osteopontin and related SIBLING glycoprotein genes are expressed by sertoli cells during mouse testis development

Matrix proteins play important roles in tissue morphogenesis. We have studied the expression of genes encoding the related SIBLING glycoproteins osteopontin (OPN), bone sialoprotein (BSP), and dentin matrix protein (DMP) during the development of male and female gonads during mouse embryogenesis. Opn mRNA was expressed specifically by Sertoli cells of the developing testis cords, in the mesonephric tubules of both sexes, and, transiently, in the Mullerian ducts of both sexes, as determined by whole-mount and section in situ hybridization. OPN protein was detected in the cytoplasm of Sertoli cells and luminal cells of the mesonephric tubules, with small amounts associated with the plasma membrane of germ cells. We found no defects in developing testes of Opn-/- mice using a range of cell type-specific markers, suggesting that other SIBLING proteins may function in testis development. Dmp and Bsp mRNA was also expressed in the developing testis cords, supporting the view that all three SIBLING proteins may contribute to testis differentiation. (c) 2005 Wiley-Liss, Inc.

The effect of copper toxicity on the growth and root morphology of Rhodes grass (Chloris gayana Knuth.) in resin buffered solution culture

A solution culture experiment was conducted to examine the effect of Cu toxicity on Rhodes grass (Chloris gayana Knuth.), a pasture species used in mine-site rehabilitation. The experiment used dilute, solution culture to achieve external nutrient concentrations, which were representative of the soil solution, and an ion exchange resin to maintain stable concentrations of Cu in solution. Copper toxicity was damaging to plant roots, with symptoms ranging from disruption of the root cuticle and reduced root hair proliferation, to severe deformation of root structure. A reduction in root growth was observed at an external Cu concentration of < 1 mu M, with damage evident from an external concentration of 0.2 mu M. Critical to the success of this experiment, in quantitatively examining the relationship between external Cu concentration and plant response, was the use of ion exchange resin to buffer the concentration of Cu in solution. After some initial difficulty with pH control, stable concentrations of Cu in solution were maintained for the major period of plant growth. The development of this technique will facilitate future investigations of the effect of heavy metals on plants.

Brain morphology in large pelagic fishes: A comparison between sharks and teleosts

A quantitative comparison was made of both relative brain size (encephalization) and the relative development of five brain area of pelagic sharks and teleosts. Two integration areas (the telencephalon and the corpus cerebellum) and three sensory brain areas (the olfactory bulbs, optic tectum and octavolateralis area, which receive primary projections from the olfactory epithelium, eye and octavolateralis senses, respectively), in four species of pelagic shark and six species of pelagic teleost were investigated. The relative proportions of the three sensory brain areas were assessed as a proportion of the total 'sensory brain', while the two integration areas were assessed relative to the sensory brain. The allometric analysis of relative brain size revealed that pelagic sharks had larger brains than pelagic teleosts. The volume of the telencephalon was significantly larger in the sharks, while the corpus cerebellum was also larger and more heavily foliated in these animals. There were also significant differences in the relative development of the sensory brain areas between the two groups, with the sharks having larger olfactory bulbs and octavolateralis areas, whilst the teleosts had larger optic tecta. Cluster analysis performed on the sensory brain areas data confirmed the differences in the composition of the sensory brain in sharks and teleosts and indicated that these two groups of pelagic fishes had evolved different sensory strategies to cope with the demands of life in the open ocean.

Definition and spatial annotation of the dynamic secretome during early kidney development

The term secretome has been defined as a set of secreted proteins (Grimmond et al. [2003] Genome Res 13:1350-1359). The term secreted protein encompasses all proteins exported from the cell including growth factors, extracellular proteinases, morphogens, and extracellular matrix molecules. Defining the genes encoding secreted proteins that change in expression during organogenesis, the dynamic secretome, is likely to point to key drivers of morphogenesis. Such secreted proteins are involved in the reciprocal interactions between the ureteric bud (UB) and the metanephric mesenchyme (AM) that occur during organogenesis of the metanephros. Some key metanephric secreted proteins have been identified, but many remain to be determined. In this study, microarray expression profiling of E10.5, E11.5, and E13.5 kidney and consensus bioinformatic analysis were used to define a dynamic secretome of early metanephric development. In situ hybridisation was used to confirm microarray results and clarify spatial expression patterns for these genes. Forty-one secreted factors were dynamically expressed between the E10.5 and E13.5 timeframe profiled, and 25 of these factors had not previously been implicated in kidney development. A text-based anatomical ontology was used to spatially annotate the expression pattern of these genes in cultured metanephric explants.

Degenerate dentition of the dugong (Dugong dugon), or why a grazer does not need teeth: morphology, occlusion and wear of mouthparts

The morphology and functional occlusion of the cheekteeth of 57 dugongs Dugong dugon of both sexes were examined using reflected light and scanning electron microscopy, radiography, hardness testing and skull manipulation. The functional morphology of the horny oral pads was also described. Mouthparts and body size allometry was examined for ontogenetic and gender-related trends. We found that the worn erupted cheekteeth of the dugong are simple flat pegs composed of soft degenerative dentine. During occlusion, the mandible moves in a mainly antero-lingual direction, with the possibility of mandibular retraction in some individuals. Anterior parts of the cheektooth row may become non-functional as a dugong ages. As a function of body size, dugong cheekteeth are extremely small compared with those of other mammalian herbivores, and with other hindgut fermenters in particular. The morphology, small size and occlusal variability of the cheekteeth suggest that there has not been strong selective pressure acting to maintain an effective dentition. In contrast, great development of the horny pads and associated skull parameters and their lower size variability suggest that the horny pads may have assumed the major role in food comminution.

Genotypic variation for cold tolerance during reproductive development in rice: Screening with cold air and cold water

Rice (Oryza sativa L.) plants are susceptible to low temperature during the young microspore stage, which occurs 10-12 days before heading. Low temperature at this time increases spikelet sterility which can cause massive yield loss. Increasing the cold tolerance of cultivars can reduce yield variability in temperate rice-growing environments. Two experiments were conducted in cold air screenings and two were conducted in cold water screenings to examine genotypic variation for cold tolerance, explore flowering traits related to spikelet sterility, and investigate whether the results reflect the level of cold tolerance determined previously in the field. Cold air screenings imposed day/night temperatures of 27 degrees C/13 degrees C, 25 degrees C/15 degrees C and 32 degrees C/25 degrees C following particle initiation until 50% heading, while cold water screenings maintained a relatively constant 19 degrees C. The variation in the commencement of low air temperature treatment did not have an effect on the level of spikelet sterility, indicating that exposure to low temperature during the young microspore stage was more important than the duration of exposure. Spikelet sterility of common cultivars showed a significant correlation between cold air and cold water screenings (r(2) = 0.63, p < 0.01), cold air and field screenings (r(2) = 0.52, p < 0.01) and cold water and field screenings (r(2) = 0.53, p < 0.01), indicating that cold air and cold water can be used for screening genotypes for low temperature tolerance. HSC55, M 103 and Jyoudeki were identified as cold tolerant and Doongara, Sasanishiki and Nipponbare as susceptible cultivars. There was a significant negative relationship between spikelet sterility and both the number of engorged pollen grains per anther and anther area only after imposing cold air and cold water treatment hence, it was concluded that these flowering traits were facultative in nature. In addition, cultivars originating from Australia and California were inefficient at producing filled grain with similar sized anthers containing a similar number of engorged pollen grains as cultivars from other origins. One suggested reason for this poor conversion to filled grain of cultivars from Australia and California may be associated with their small stigma area, particularly when exposed to low temperature conditions. (c) 2006 Elsevier B.V. All rights reserved.

Minimising cold damage during reproductive development among temperate rice genotypes. II. Genotypic variation and flowering traits related to cold tolerance screening

Low temperature during microspore development increases spikelet sterility and reduces grain yield in rice (Oryza sativa L.). The objectives of this study were to determine genotypic variation in spikelet sterility in the field in response to low-temperature and then to examine the use of physio-morphological traits at flowering to screen for cold tolerance. Multiple-sown field experiments were conducted over 4 consecutive years in the rice-growing region of Australia to increase the likelihood of encountering low-temperature during microspore development. More than 50 cultivars of various origins were evaluated, with 7 cultivars common to all 4 years. The average minimum temperature for 9 days during microspore development was used as a covariate in the analysis to compare cultivars at a similar temperature. The low-temperature conditions in Year 4 identified cold-tolerant cultivars such as Hayayuki and HSC55 and susceptible cultivars such as Sasanishiki and Doongara. After low temperature conditions, spikelet sterility was negatively correlated with the number of engorged pollen grains, anther length, anther area, anther width, and stigma area. The number of engorged pollen grains and anther length were found to be facultative traits as their relationships with spikelet sterility were identified only after cold water exposure and did not exist under non-stressed conditions.

The number, morphology, and distribution of retinal ganglion cells and optic axons in the Australian lungfish Neoceratodus forsteri (Krefft 1870)

Australian lungfish Neoceratodus forsteri may be the closest living relative to the first tetrapods and yet little is known about their retinal ganglion cells. This study reveals that lungfish possess a heterogeneous population of ganglion cells distributed in a horizontal streak across the retinal meridian, which is formed early in development and maintained through to adult stages. The number and complement of both ganglion cells and a population of putative amacrine cells within the ganglion cell layer are examined using retrograde labelling from the optic nerve and transmission electron-microscopic analysis of axons within the optic nerve. At least four types of retinal ganglion cells are present and lie predominantly within a thin ganglion cell layer, although two subpopulations are identified, one within the inner plexiform and the other within the inner nuclear layer. A subpopulation of retinal ganglion cells comprising up to 7% or the total population are significantly larger (> 400 mu m(2)) and are characterized as giant or alpha-like cells. Up to 44% of cells within the retinal ganglion cell layer represent a population of presumed amacrine cells. The optic nerve is heavily fasciculated and the proportion of myelinated axons increases with body length from 17% in subadults to 74% in adults. Spatial resolving power, based on ganglion cell spacing, is low (1.6-1.9 cycles deg(-1), n = 2) and does not significantly increase with growth. This represents the first detailed study of retinal ganglion cells in sarcopterygian fish, and reveals that, despite variation amongst animal groups, trends in ganglion cell density distribution and characteristics of cell types were defined early in vertebrate evolution.

Low dietary protein during early pregnancy alters bovine placental development

To determine if low dietary protein concentration in the first two trimesters of pregnancy alters placental development, genetically similar heifers from closed herd were fed diets containing different levels of protein in the first and second trimesters of gestation. There were four animals per treatment group, the groups being: L/L = fed a diet containing 7% crude protein (CP) (low protein) in the first and second trimesters; H/H = fed a diet containing 14% (P thigh protein) in the first and second trimesters; L/H = fed low protein in the first trimester and high in the second trimester and vice versa for the H/L group. Low protein diets in the first trimester increased dry cotyledon weight at term. Trophectoderm volume density increased in the H/L and L/H group compared to the L/L and H/H groups. Blood vessel volume and volume density in foetal villi decreased in the H/L and L/H groups compared with the H/H and L/L groups. There was no effect of diet treatment on cotyledon number, diameter or wet weight and no effect on the volume density of connective tissue or fibroblasts in the foetal villi. These results show that a low dietary protein concentration in the first trimester of pregnancy followed by increased protein in the second trimester enhanced placental development. Further, trophectoderm volume was highly correlated with birth weight. Early protein restriction in the pregnant cow may enhance foetal growth in part by stimulating placental growth and function. (C) 1999 Published by Elsevier Science B.V. All rights reserved.

Modelling broccoli development, yield and quality

Broccoli is a vegetable crop of increasing importance in Australia, particularly in south-east Queensland and farmers need to maintain a regular supply of good quality broccoli to meet the expanding market. A predictive model of ontogeny, incorporating climatic data including frost risk, would enable farmers to predict harvest maturity date and select appropriate cultivar – sowing date combinations. To develop procedures for predicting ontogeny, yield and quality, field studies using three cultivars, ‘Fiesta’, ‘Greenbelt’ and ‘Marathon’, were sown on eight dates from 11 March to 22 May 1997, and grown under natural and extended (16 h) photoperiods at the University of Queensland, Gatton Campus. Cultivar, rather than the environment, mainly determined head quality attributes of head shape and branching angle. Yield and quality were not influenced by photoperiod. A better understanding of genotype and environmental interactions will help farmers optimise yield and quality, by matching cultivars with time of sowing. The estimated base and optimum temperature for broccoli development were 0°C and 20 °C, respectively, and were consistent across cultivars, but thermal time requirements for phenological intervals were cultivar specific. Differences in thermal time requirement from floral initiation to harvest maturity between cultivars were small and of little importance, but differences in thermal time requirement from emergence to floral initiation were large. Sensitivity to photoperiod and solar radiation was low in the three cultivars used. This research has produced models to assist broccoli farmers in crop scheduling and cultivar selection in south-east Queensland.

The effect of ageing on in vivo human ciliary muscle morphology and contractility

Purpose. To assess the effect of ageing on in vivo human ciliary muscle morphology and contractility during accommodation. Methods. Seventy-nine subjects, aged 19–70 years were recruited. High-resolution images were acquired of nasal and temporal ciliary muscle in the relaxed state, and at stimulus vergence levels of -4 and -8 D, using anterior segment optical coherence tomography (AS-OCT). Objective refractions and axial lengths were also recorded. Linear regression analysis was performed to determine the effect of age on nasal and temporal ciliary muscle morphologic characteristics. Results. Ciliary muscle anterior length decreased significantly with age both nasally (R = 0.461, P = 0.001) and temporally (R = 0.619, P < 0.001) in emmetropic eyes. In a subset of 37 participants, ciliary muscle maximum width increased significantly with age, by 2.8 µm/year nasally (R = 0.54, P < 0.001) and 3.0 µm/year temporally (R = 0.44, P = 0.007), while the distance from the inner apex of the ciliary muscle to the scleral spur decreased significantly with age on both the nasal and temporal aspects (R = 0.47; P = 0.004 and R = 0.43; P = 0.009, respectively). During accommodation, changes to ciliary muscle thickness and length remained constant throughout life. Conclusions. The human ciliary muscle undergoes age-dependent changes in morphology that suggest an antero-inwards displacement of muscle mass, particularly in emmetropic eyes. However, the morphologic changes observed appear not to affect the ability of the muscle to contract during accommodation, even in established presbyopes, thus supporting a lenticular model of presbyopia development.

Factors determining the morphology of β-amyloid (Aβ) deposits in Down's syndrome

Immunostained preparations of the medial temporal lobe from patients with Down's syndrome (DS) were counterstained with cresyl violet to reveal the β-amyloid (Aβ) deposits and their associated cell populations. Aβ deposits in the cornu Ammonis (CA) of the hippocampus were, on average, more strongly stained, less often directly associated with neurons and more often associated with glial cells than the adjacent areas of cortex. Cored deposits were more frequently recorded in sulci rather than gyri and were associated with more glial cells than the uncored deposits. Multiple regression analyses suggested there was a positive correlation in the cortex between Aβ deposit size and the frequency of closely associated neurons, the correlation being most significant with larger (>25 μm) neurons. The morphology of Aβ deposit was also correlated with the location of deposits in the cortex, CA and dentate gyrus but this factor was of lesser importance. No significant variation in the morphology of the Aβ deposits was associated with the presence of blood vessels within or adjacent to the deposit. The data suggest that neuronal cell bodies are important in the initial formation of Aβ deposits and glial cells with the development of more mature amyloid deposits.

Development of plasmid dna formulations for application as microspheric dna vaccines

The advent of DNA vaccines has heralded a new technology allowing the design and elicitation of immune responses more adequate for a wider range of pathogens. The formulation of these vaccines into the desired dosage forms extends their capability in terms of stability, routes of administration and efficacy. This thesis describes an investigation into the fabrication of plasmid DNA, the active principle of DNA vaccines, into microspheres, based on the tenet of an increased cellular uptake of microparticulate matter by phagocytic cells. The formulation of plasmid DNA into microspheres using two methods, is presented. Formulation of microspheric plasmid DNA using the double emulsion solvent evaporation method and a spray-drying method was explored. The former approach involves formation of a double emulsion, by homogenisation. This method produced microspheres of uniform size and smooth morphology, but had a detrimental effect on the formulated DNA. The spray-drying method resulted in microspheres with an improved preservation of DNA stability. The use of polyethylenimine (PEI) and stearylamine (SA) as agents in the microspheric formulation of plasmid DNA is a novel approach to DNA vaccine design. Using these molecules as model positively-charged agents, their influence on the characteristics of the microspheric formulations was investigated. PEI improved the entrapment efficiency of the plasmid DNA in microspheres, and has minimal effect on either the surface charge, morphology or size distribution of the formulations. Stearylamine effected an increase in the entrapment efficiency and stability of the plasmid DNA and its effect on the micropshere morphology was dependent on the method of preparation. The differences in the effects of the two molecules on microsphere formulations may be attributable to their dissimilar physico-chemical properties. PEI is water-soluble and highly-branched, while SA is hydrophobic and amphipathic. The positive charge of both molecules is imparted by amine functional groups. Preliminary data on the in vivo application of formulated DNA vaccine, using hepatitis B plasmid, showed superior humoral responses to the formulated antigen, compared with free (unformulated) antigen.

Development of visual evoked responses to tritan,red-green and luminance stimuli in human infants

The principal aim of this work was to investigate the development of the S-cone colour-opponent pathway in human infants aged 4 weeks to 6 months. This was achieved by recording transient visual evoked responses to pattern-onset stimuli along a tritanopic confusion axis (tritan stimuli) at and around the adult isoluminant match. For comparison, visual evoked responses to red-green and luminance-modulated stimuli were recorded from the same infants at the same ages. Evoked responses were also recorded from colour-normal adults for comparison with those of the infants. The transient VEP allowed observation of response morphology as luminance differences were introduced to the chromatic stimuli. In this way, an estimate of isoluminance was possible in infants. Estimated isoluminant points for a group of six infants aged 6 to 10 weeks closely approximated the adult isoluminant match. This finding has implications for the use of photometric isoluminance in infant work, and suggests that photopic spectral sensitivity is similar in infants and adults. Abnormalities of the visual evoked responses to tritan, red-green and luminance-modulated stimuli in an infant with cystic fibrosis are reported. The results suggest abnormal function of the retino-striate visual pathway in this infant, and it is argued that these may be secondary to his illness, although data from more infants with cystic fibrosis are needed to clarify this further. A group of nine healthy infants demonstrated evoked responses to tritan stimuli by 4 to 10 weeks and to red-green stimuli by 6 to 11 weeks post-term age. Responses to luminance-modulated stimuli were present in all nine infants at the earliest age tested, namely 4 weeks post-term. The slightly earlier age of onset of evoked responses to tritan stimuli than for red-green may be explained by the relatively lower cone contrast afforded by red-green stimuli. Latency of the evoked response to both types of chromatic stimuli and to luminance-modulated stimuli decreased with age at a similar rate, suggesting that the visual pathways transmitting luminance and chromatic information mature at similar rates in young infants.

Development of a co-culture model of the human lungs for toxicity testing and identification of biomarkers of inhalation toxicity

The airway epithelium is the first point of contact in the lung for inhaled material, including infectious pathogens and particulate matter, and protects against toxicity from these substances by trapping and clearance via the mucociliary escalator, presence of a protective barrier with tight junctions and initiation of a local inflammatory response. The inflammatory response involves recruitment of phagocytic cells to neutralise and remove and invading materials and is oftern modelled using rodents. However, development of valid in vitro airway epithelial models is of great importance due to the restrictions on animal studies for cosmetic compound testing implicit in the 7th amendment to the European Union Cosmetics Directive. Further, rodent innate immune responses have fundamental differences to human. Pulmonary endothelial cells and leukocytes are also involved in the innate response initiated during pulmonary inflammation. Co-culture models of the airways, in particular where epithelial cells are cultured at air liquid interface with the presence of tight junctions and differentiated mucociliary cells, offer a solution to this problem. Ideally validated models will allow for detection of early biomarkers of response to exposure and investigation into inflammatory response during exposure. This thesis describes the approaches taken towards developing an in vitro epithelial/endothelial cell model of the human airways and identification biomarkers of response to exposure to xenobiotics. The model comprised normal human primary microvascular endothelial cells and the bronchial epithelial cell line BEAS-2B or normal human bronchial epithelial cells. BEAS-2B were chosen as their characterisation at air liquid interface is limited but they are robust in culture, thereby predicted to provide a more reliable test system. Proteomics analysis was undertaken on challenged cells to investigate biomarkers of exposure. BEAS-2B morphology was characterised at air liquid interface compared with normal human bronchial epithelial cells. The results indicate that BEAS-2B cells at an air liquid interface form tight junctions as shown by expression of the tight junction protein zonula occludens-1. To this author’s knowledge this is the first time this result has been reported. The inflammatory response of BEAS-2B (measured as secretion of the inflammatory mediators interleukin-8 and -6) air liquid interface mono-cultures to Escherichia coli lipopolysaccharide or particulate matter (fine and ultrafine titanium dioxide) was comparable to published data for epithelial cells. Cells were also exposed to polymers of “commercial interest” which were in the nanoparticle range (and referred to particles hereafter). BEAS-2B mono-cultures showed an increased secretion of inflammatory mediators after challenge. Inclusion of microvascular endothelial cells resulted in protection against LPS- and particle- induced epithelial toxicity, measured as cell viability and inflammatory response, indicating the importance of co-cultures for investigations into toxicity. Two-dimensional proteomic analysis of lysates from particle-challenged cells failed to identify biomarkers of toxicity due to assay interference and experimental variability. Separately, decreased plasma concentrations of serine protease inhibitors, and the negative acute phase proteins transthyretin, histidine-rich glycoprotein and alpha2-HS glycoprotein were identified as potential biomarkers of methyl methacrylate/ethyl methacrylate/butylacrylate treatment in rats.