The distribution and morphological characteristics of cholinergic cells in the brain of monotremes as revealed by ChAT immunohistochemistry

The present study employs choline acetyltransferase (ChAT) immunohistochemistry to identify the cholinergic neuronal population in the central nervous system of the monotremes. Two of the three extant species of monotreme were studied: the platypus (Omithorhynchus anatinus) and the short-beaked echidna (Tachyglossus aculeatus). The distribution of cholinergic cells in the brain of these two species was virtually identical. Distinct groups of cholinergic cells were observed in the striatum, basal forebrain, habenula, pontomesencephalon, cranial nerve motor nuclei, and spinal cord. In contrast to other tetrapods studied with this technique, we failed to find evidence for cholinergic cells in the hypothalamus, the parabigeminal nucleus (or nucleus isthmus), or the cerebral cortex. The lack of hypothalamic cholinergic neurons creates a hiatus in the continuous antero-posterior aggregation of cholinergic neurons seen in other tetrapods. This hiatus might be functionally related to the phenomenology of monotreme sleep and to the ontogeny of sleep in mammals, as juvenile placental mammals exhibit a similar combination of sleep elements to that found in adult monotremes. Copyright (C) 2002 S. Karger AG, Basel.

Distribution of two splice variants of the glutamate transporter GLT1 in the retinas of humans, monkeys, rabbits, rats, cats, and chickens

Antibodies have been generated against two carboxyl-terminal splice variants of the glutamate transporter GLT1, namely, the originally described version of GLT1 and GLT1-B, and labelling has been examined in multiple species, including chickens and humans. Although strong specific labelling was observed in each species, divergent patterns of expression were noted. Moreover, each antibody was sensitive to the phosphorylation state of the appropriate protein, because chemical removal of phosphates using alkaline phosphatase revealed a broader range of labelled elements in most cases. In general, GLT1-B was present in cone photoreceptors and in rod and cone bipolar cells in the retinas of rabbits, rats, and cats. In the cone-dominated retinas of chickens and in marmosets, GLT1-B was associated only with cone photoreceptors, whereas, in macaque and human retinas, GLT1-B was associated with bipolar cells and terminals of photoreceptors. In some species, such as cats, GLT-B was also present in horizontal cells. By contrast, GLT1 distribution varied. GLT1 was associated with amacrine cells in chickens, rats, cats, and rabbits and with bipolar cells in marmosets and macaques. In the rat retina, rod photoreceptor terminals also contained GLT1, but this was evident only in enzymatically dephosphorylated tissues. We conclude that the two variants of GLT1 are present in all species examined but are differentially distributed in a species-specific manner. Moreover, each cell type generally expresses only one splice variant of GLT1. J. Comp. Neurol. 445:1-12, 2002. (C) 2002 Wiley-Liss, Inc.

Distribution of two splice variants of the glutamate transporter GLT-1 in rat brain and pituitary

We have performed immunocytochemistry on rat brains using a highly specific antiserum directed against the originally described form of the glutamate transporter GLT-1 (referred to hereafter as GLT-1alpha), and another against a C-terminal splice variant of this protein, GLT-1B. Both forms of GLT-1 were abundant in rat brain, especially in regions such as the hippocampus and cerebral cortex, and macroscopic examination of sections suggested that both forms were generally regionally coexistent. However, disparities were evident; GLT-1alpha was present in the intermediate lobe of the pituitary gland, whereas GLT-1B was absent. Similar marked disparities were also noted in the external capsule, where GLT1A labeling was abundant but GLT-1B was only occasionally encountered. Conversely, GLT-1B was more extensively distributed, relative to GLT-1alpha, in areas such as the deep cerebellar nuclei. In most regions, such as the olfactory bulbs, both splice variants were present but differences were evident in their distribution. In cerebral cortex, patches were evident where GLT-1B was absent, whereas no such patches were evident for GLT-1alpha. At high resolution, other discrepancies were evident; double-labeling of areas such as hippocampus indicated that the. two splice variants may either be differentially expressed by closely apposed glial elements or that the two splice variants may be differentially targeted to distinct membrane domains of individual glial cells. (C) 2002 Wiley-Liss, Inc.

Distribution of two splice variants of the glutamate transporter GLT-1 in the developing rat retina

The distributions of a carboxyl terminal splice variant of the glutamate transporter GLT-1, referred to as GLT-1B, and the carboxyl terminus of the originally described variant of GLT-1, referred to hereafter as GLT-1alpha, were examined using specific antisera. GLT-1B was present in the retina at very early developmental stages. Labelling was demonstrable at embryonic day 14, and strong labelling was evident by embryonic day 18. Such labelling was initially restricted to populations of cone photoreceptors, the processes of which extended through the entire thickness of the retina and appeared to make contact with the retinal ganglion cells. During postnatal development the GLT-1B-positive photoreceptor processes retracted to form the outer plexiform layer, and around postnatal day 7, GLT-1B-immunoreactive bipolar cells appeared. The pattern of labelling of bipolar cell processes within the inner plexiform layer changed during postnatal development. Two strata of strongly immunoreactive terminals were initially evident in the inner plexiform layer, but by adulthood these two bands were no longer evident and labelling was restricted to the somata and processes (but not synaptic terminals) of the bipolar cells, as well as the somata, processes, and terminals of cone photoreceptors. By contrast, GLT-1alpha appeared late in postnatal development and was restricted mainly to a population of amacrine cells, although transient labelling was also associated with punctate elements in the outer plexiform layer, which may represent photoreceptor terminals, (C) 2002 Wiley-Liss, Inc.

Effects of habitat characteristics and climate on the distribution and colouration of Dusky Moorhens (Gallinula tenebrosa) in south-east Queensland

We determined which factors predict the presence and abundance of Dusky Moorhens (Gallinula tenebrosa) at wetlands by surveying the ecological and habitat characteristics of 62 sites across south-east Queensland. Moorhens were observed in 48 of the sites sampled. They were more likely to be found at sites surrounded by taller terrestrial vegetation and where free-floating and attached aquatic vegetation was more abundant. The number of moorhens found at a site increased in relation to vegetation height, the abundance of attached aquatic vegetation and the number of purple swamphens observed. These results suggest that there are ecological constraints on the distribution of moorhens, and that food abundance and the availability of suitable nesting sites determine the overall distribution and abundance of moorhens in wetlands. Adult moorhens develop brightly coloured fleshy frontal shields, bills and legs when breeding, although in some populations birds maintain year-round colouration. We observed year-round breeding colouration in 23 out of 34 sampling sites that had moorhens and were surveyed in August. Coloured moorhens were found during winter at sites with higher minimum winter temperatures, and more abundant free-floating and submerged leafy vegetation. In addition, higher proportions of moorhens were coloured at sites with higher mean minimum temperatures. The retention of year-round breeding colouration appears to be restricted to areas with warmer winter temperatures and more abundant food. The results suggest that areas not occupied by moorhens are of inadequate quality to support breeding populations. We suggest that ecological constraints on independent breeding in Dusky Moorhens may have favoured the evolution of their unusual cooperative breeding system, which involves frequent mate-sharing by both sexes.

Overwintering, soil distribution and phenology of Childers canegrub, Antitrogus parvulus (Coleoptera : Scarabaeidae) in Queensland sugarcane

In this study, the question of whether Childers canegrub, Antitrogus parvulus (Britton) overwinters in the subsoil was addressed. Irrigated fields of sugarcane were sampled during a 2-year period near Bundaberg in southern Queensland. Antitrogus parvulus overwintered as second and third instars at each of three sites. During autumn and winter third instars of different allochronic (separated in age by 12 months) populations occurred together and could not be readily separated. Field-collected third instars were reared on ryegrass and separated into two age groups based on the date of pupation. Third instars in the first year of their life cycle (young third instars) remained at shallow depth (100-200 mm) and did not overwinter in the subsoil as once thought. Minimum temperatures during winter were 13-16degreesC and did not prevent young third instars from feeding and gaining weight. Third instars in their second and final year moved downwards from late summer and pupated in the subsoil at 293-425 mm in spring. General phenology was as previously reported with first instar larvae occurring from January until April, second instars from January until November and third instar larvae throughout the year. Prepupae and pupae were found between October and December and adults occurred in soil during November and January. Batches of eggs occurred at a mean depth of 350 mm. First and second instars occurred predominantly at relatively shallow (100-200 mm) depths in the soil profile. All stages tended to be most common under rows of sugarcane rather than in the interrow.

Immunohistochemical localization of fibromodulin in the periodontium during cementogenesis and root formation in the rat molar

Background: Cementum is essential for periodontal regeneration, as it provides anchorage between the root surface and the periodontal ligament. A variety of macromolecules present in the extracellular matrix of the periodontium, including proteoglycans, are likely to play a regulatory role in cementogenesis. Recently, the small leucine-rich proteoglycan, fibromodulin, has been isolated from bovine periodontal ligament and localized in bovine cementum, as well as in human periodontal ligament. Objective: The aim of this study was to examine the distribution of fibromodulin during cementogenesis and root formation. Methods: A standard indirect immunoperoxidase technique was employed, using an antifibromodulin polyclonal antibody on sections of molar teeth from rats aged 3, 5 and 8 weeks. Results: Immunoreactivity to fibromodulin was evident in the periodontal ligament in all sections. An intense positive stain was observed in the extracellular matrix where the periodontal ligament fibers insert into the alveolar bone and where the Sharpey's fibers insert into the cementum. There was no staining evident in the mineralized cellular and acellular cementum. The intensity of immunoreactivity to the antifibromodulin antibody increased proportionally with increasing tissue maturation. Conclusion: The results from this study suggest that fibromodulin is a significant component of the extracellular matrix in the periodontal ligament during development, and may play a regulatory role in the mineralization process or maintaining homeostasis at the hard-soft tissue interface during cementogenesis.

Distribution and abundance of dugongs in Moreton Bay, Queensland, Australia

Dugong abundances in Moreton Bay (south-east Queensland) were estimated during six bi- monthly aerial surveys throughout 1995. Sampling intensity ranged between 20 and 80% for different sampling zones within the Bay, with a mean intensity of 40.5%. Population estimates for dugongs were corrected for perception bias ( the proportion of animals visible in the transect that were missed by observers), and standardised for availability bias ( the proportion of animals that were invisible due to water turbidity) with survey and species-specific correction factors. Population estimates for dugongs in Moreton Bay ranged from 503 +/- 64 (s.e.) in July to 1019 +/- 166 in January. The highest uncorrected count was 857 dugongs in December. This is greater than previous population estimates, suggesting that either previous surveys have underestimated abundance and/or that this population may have increased through recruitment, immigration, or a combination of both. The high degree of variation in population estimates between surveys may be due to temporal differences in distribution and herding behaviour. In winter, dugongs were found in smaller herds and were dispersed over a wider area than in summer. The Eastern Banks region of the bay supported 80 - 98% of the dugong population at any one time. Within this region, there were several dugong 'hot spots' that were visited repeatedly by large herds. These 'hot spots' contained seagrass communities that were dominated by species that dugongs prefer to eat. The waters of Rous Channel, South Passage and nearby oceanic waters are also frequently inhabited by dugongs in the winter months. Dugongs in other parts of Moreton Bay were at much lower densities than on the Eastern Banks.

C-terminal sequences in R-Ras are involved in integrin regulation and in plasma membrane microdomain distribution

The small GTPases R-Ras and H-Ras are highly homologous proteins with contrasting biological properties, for example, they differentially modulate integrin affinity: H-Ras suppresses integrin activation in fibroblasts whereas R-Ras can reverse this effect of H-Ras. To gain insight into the sequences directing this divergent phenotype, we investigated a panel of H-Ras/R-Ras chimeras and found that sequences in the R-Ras hypervariable C-terminal region including amino acids 175-203 are required for the R-Ras ability to increase integrin activation in CHO cells; however, the proline-rich site in this region, previously reported to bind the adaptor protein Nck, was not essential for this effect. In addition, we found that the GTPase TC21 behaved similarly to R-Ras. Because the C-termini of Ras proteins can control their subcellular localization, we compared the localization of H-Ras and R-Ras. In contrast to H-Ras, which migrates out of lipid rafts upon activation, we found that activated R-Ras remained localized to lipid rafts. However, functionally distinct H-Ras/R-Ras chimeras containing different C-terminal R-Ras segments localized to lipid rafts irrespective of their integrin phenotype. (C) 2003 Elsevier Inc. All rights reserved.

Low-density koala (Phascolarctos cinereus) populations in the mulgalands of south-west Queensland. II. Distribution and diet

This study used faecal pellets to investigate the broadscale distribution and diet of koalas in the mulgalands biogeographic region of south-west Queensland. Koala distribution was determined by conducting faecal pellet searches within a 30-cm radius of the base of eucalypts on 149 belt transects, located using a multi-scaled stratified sampling design. Cuticular analysis of pellets collected from 22 of these sites was conducted to identify the dietary composition of koalas within the region. Our data suggest that koala distribution is concentrated in the northern and more easterly regions of the study area, and appears to be strongly linked with annual rainfall. Over 50% of our koala records were obtained from non-riverine communities, indicating that koalas in the study area are not primarily restricted to riverine communities, as has frequently been suggested. Cuticular analysis indicates that more than 90% of koala diet within the region consists of five eucalypt species. Our data highlights the importance of residual Tertiary landforms to koala conservation in the region.

Tissue engineering for bone regeneration using differentiated alveolar bone cells in collagen scaffolds

Regeneration of osseous defects by a tissue-engineering approach provides a novel means of treatment utilizing cell biology, materials science, and molecular biology. In this study the concept of tissue engineering was tested with collagen type I matrices seeded with cells with osteogenic potential and implanted into sites where osseous damage had occurred. Explant cultures of cells from human alveolar bone and gingiva were established. When seeded into a three-dimensional type I collagen-based scaffold, the bone-derived cells maintained their osteoblastic phenotype as monitored by mRNA and protein levels of the bone-related proteins including bone sialoprotein, osteocalcin, osteopontin, bone morphogenetic proteins 2 and 4, and alkaline phosphatase. These in vitro-developed matrices were implanted into critical-size bone defects in skulls of immunodeficient (SCID) mice. Wound healing was monitored for up to 4 weeks. When measured by microdensitometry the bone density within defects filled with osteoblast-derived matrix was significantly higher compared with defects filled with either collagen scaffold alone or collagen scaffold impregnated with gingival fibroblasts. New bone formation was found at all the sites treated with the osteoblast-derived matrix at 28 days, whereas no obvious new bone formation was identified at the same time point in the control groups. In situ hybridization for the human-specific Alu gene sequence indicated that the newly formed bone tissue resulted from both transplanted human osteoblasts and endogenous mesenchymal stem cells. The results indicate that cells derived from human alveolar bone can be incorporated into bioengineered scaffolds and synthesize a matrix, which on implantation can induce new bone formation.

A foliar rating system for comparing the resistance of banana cultivars grown as tissue-cultured plantlets in the laboratory to Fusarium wilt

A foliar rating system was developed to assess the progress of Fusarium wilt ( Panama disease) caused by Fusarium oxysporum f. sp. cubense in seven banana cultivars differing in their resistance to race 1 of the pathogen. Plantlets were transplanted into unamended soil naturally infested with the pathogen, soil amended with urea and soil amended with aged chicken manure. A corm invasion score was also developed to assess the accuracy of the foliar symptom score as an indicator of cultivar resistance. On the basis of foliar symptom scores alone, the response of five of the seven cultivars in the chicken manure treatment corresponded to their known field response. However, the response of the other two cultivars, both susceptible to the pathogen in the field, fell into two categories. One had a high foliar symptom score and a correspondingly high corm invasion score, whereas the other had a low foliar symptom score and a high corm invasion score. Breeders need to be aware of the two categories of susceptible response, if inferior breeding material is to be rejected early on in a breeding program.

Calcium treatment of harvested Geraldton waxflower does not enhance postharvest quality

Postharvest flower abscission from cut Geraldton waxflower (Chamelaucium uncinatum) is mostly caused by fungal invasion. Elevated plant tissue calcium concentrations through postharvest application reduces fungal disease severity in various crops. Such results may be explained by strengthening of plant cell walls by calcium. Strengthening provides a structural barrier to fungal hyphae, thereby restricting invasion of plant cells. Postharvest pulsing with calcium solution substantially increased calcium concentrations in waxflower tissues. Ca-45 tracer revealed calcium distribution throughout flowering sprigs, including infection sites such as stylar tissue. However, pulsing waxflower sprigs with calcium did not suppress either disease or flower abscission, nor did it enhance vase life.

Biochemical and molecular studies using human autopsy brain tissue

The use of human brain tissue obtained at autopsy for neurochemical, pharmacological and physiological analyses is reviewed. RNA and protein samples have been found suitable for expression profiling by techniques that include RT-PCR, cDNA microarrays, western blotting, immunohistochemistry and proteomics. The rapid development of molecular biological techniques has increased the impetus for this work to be applied to studies of brain disease. It has been shown that most nucleic acids and proteins are reasonably stable post-mortem. However, their abundance and integrity can exhibit marked intra- and intercase variability, making comparisons between case-groups difficult. Variability can reveal important functional and biochemical information. The correct interpretation of neurochemical data must take into account such factors as age, gender, ethnicity, medicative history, immediate ante-mortem status, agonal state and post-mortem and post-autopsy intervals. Here we consider issues associated with the sampling of DNA, RNA and proteins using human autopsy brain tissue in relation to various ante- and post-mortem factors. We conclude that valid and practical measures of a variety of parameters may be made in human brain tissue, provided that specific factors are controlled.

Distribution of Chlamydia pneumoniae DNA in atherosclerotic carotid arteries: significance for sampling procedures

Despite extensive efforts to confirm a direct association between Chlamydia pneumoniae and atherosclerosis, different laboratories continue to report a large variability in detection rates. In this study, we analyzed multiple sections from atherosclerotic carotid arteries from 10 endartectomy patients to determine the location of C. pneumoniae DNA and the number of sections of the plaque required for analysis to obtain a 95% confidence of detecting the bacterium. A sensitive nested PCR assay detected C. pneumoniae DNA in all patients at one or more locations within the plaque. On average, 42% (ranging from 5 to 91%) of the sections from any single patient had C. pneumoniae DNA present. A patchy distribution of C. pneumoniae in the atherosclerotic lesions was observed, with no area of the carotid having significantly more C. pneumoniae DNA present. If a single random 30-mum-thick section was tested, there was only a 35.6 to 41.6% (95% confidence interval) chance of detecting C. pneumoniae DNA in a patient with carotid artery disease. A minimum of 15 sections would therefore be required to obtain a 95% chance of detecting all true positives. The low concentration and patchy distribution of C. pneumoniae DNA in atherosclerotic plaque appear to be among the reasons for inconsistency between laboratories in the results reported.