Genome size variation in deep-sea amphipods.

Genome size varies considerably across taxa, and extensive research effort has gone into understanding whether variation can be explained by differences in key ecological and life-history traits among species. The extreme environmental conditions that characterize the deep sea have been hypothesized to promote large genome sizes in eukaryotes. Here we test this supposition by examining genome sizes among 13 species of deep-sea amphipods from the Mariana, Kermadec and New Hebrides trenches. Genome sizes were estimated using flow cytometry and found to vary nine-fold, ranging from 4.06 pg (4.04 Gb) in Paralicella caperesca to 34.79 pg (34.02 Gb) in Alicella gigantea. Phylogenetic independent contrast analysis identified a relationship between genome size and maximum body size, though this was largely driven by those species that display size gigantism. There was a distinct shift in the genome size trait diversification rate in the supergiant amphipod A. gigantea relative to the rest of the group. The variation in genome size observed is striking and argues against genome size being driven by a common evolutionary history, ecological niche and life-history strategy in deep-sea amphipods.

Improving quantification using curtain flow chromatography columns in the analysis of labile compounds: a study on amino acids.

The performance of curtain flow chromatography column technology with MS detection was evaluated for the analysis of labile compounds. The curtain flow column design allows for separations that are faster and/or more sensitive than conventional columns, depending on how exactly the curtain flow column is configured. For example, when mass spectral detection is employed, the curtain flow column can yield separations that are 5-times faster than conventional columns when the curtain flow and the conventional columns have the same internal diameter. Or when the internal diameter of the conventional column is reduced in order to yield the same analytical through-put as the curtain flow column, the sensitivity on the curtain flow column can be as much as 66-fold higher than the conventional column. As a consequence of the higher analytical through-put less standardization is required in the analysis of labile compounds because less sample degradation is apparent. Consequently the sample integrity is preserved yielding data of a higher quality.

High through-put and highly sensitive liquid chromatography-tandem mass spectrometry separations of essential amino acids using active flow technology chromatography columns.

A preliminary investigation was undertaken to assess the performance of a new chromatography column technology in applications involving liquid chromatography coupled to mass spectrometry. The new column design allows mobile phase and solute to be extracted from the radial central region of the column, which reduces the solvent load to the mass spectrometer and improves separation efficiency. Effectively the column functions as a 'wall-less' column. The advantages of this design is that the analysis through-put can be increased by a factor of five, while at the same time there is a reduction in baseline noise, which results in an increase in the signal to noise response by up to 10-fold in comparison to standard columns with the same internal diameter and approaching 66-fold in comparison to standard columns with the same virtual internal diameter.

Reaction flow chromatography for rapid post column derivatisations: the analysis of antioxidants in natural products.

The analysis of antioxidants from complex samples is conveniently achieved using liquid chromatography, which provides sample fraction, coupled with an on-line antioxidant assay, which provides detection. One particularly useful on-line antioxidant assay that has routinely been coupled with HPLC involves the diphenylpicrylhydrazyl radical (DPPH), which provides a positive test for phenolic antioxidants through a decolorisation of the DPPH reagent. A limitation of this assay, however, is the need to employ a reaction coil, which is often large with respect to the peak volume, consequently adding substantial band broadening to the separation. In this study we introduce a new concept that can be employed for systems requiring post column derivatisations, such as the DPPH assay. We have termed this 'reaction flow' chromatography, whereby, the derivatisation reagent can be added directly into one of the outlet ports of a parallel segmented flow column. Subsequently, the mixing between the derivatising reagent and the solute is very efficient removing the need to employ reaction coils. The concept is tested here using the DPPH assay for the analysis of antioxidants in samples derived from natural origin.

Gradient elution chromatography with segmented parallel flow column technology: a study on 4.6 mm analytical scale columns.

A new column format known as parallel segmented flow has recently been introduced, whereby improvements in column performance are observed. These improvements are achieved via the separation of eluent from the column core from that of the column wall region. The segmentation of flow is accomplished immediately as the eluent exits the column through the use of a multi-channel end fitting. The ratio of flow exiting through the column central port relative to the peripheral ports, known as the segmentation ratio, can be tuned to optimise chromatographic performance. Investigations into the use of parallel segmented flow chromatography columns have demonstrated increased sensitivity and theoretical plates in analytical scale isocratic separations, but so far no studies have detailed the performance of these columns in gradient elution. The current study addresses the performance of parallel segmented flow columns in gradient elution, detailing the reproducibility of the gradient at various segmentation ratios and compares the performance to conventional columns. The study found that there was no observable difference in the gradient shape, or reproducibility of the gradient profiles generated at any segmentation ratio, tested on three different types of stationary phases. A separation of an 11-component test mixture verified that the primary advantage of parallel segmented flow columns was that the peak volume was reduced in proportion to the segmentation ratio.

Parallel segmented flow chromatography columns: conventional analytical scale column formats presenting as a 'virtual' narrow bore column.

Narrow bore columns find advantage in HPLC applications when volumetric flow is important, For example, for detection processes that are volume limited. Yet there are significant drawbacks to narrow bore columns. Due to their small column volume relative to analytical scale columns, narrow bore columns are more affected by system dead volume. In addition the wall effect and the variation in packing density from the centre to the wall are more significant in these columns relative to larger scale analytical columns. In this study we operate a 4.6mm i.d. parallel segmented flow column in such a manner that it emulates 2.1mm i.d. and 3.0mm i.d. columns. By using a parallel segmented flow column in this way, it was possible to combine the benefits of narrow bore and analytical scale columns.

Antipsychotic drugs cause bradycardia in GD 13 rat embryos in vitro.

This study investigated the effects of antipsychotic drugs on heart function of gestational day (GD) 13 rat embryos in vitro since they all block the I(Kr)/hERG potassium ion channel in addition to their main pharmacological effect on neurotransmitters. The results showed that all the tested antipsychotic drugs caused bradycardia of the rat embryonic heart in a concentration-dependent manner. However, with the possible exception of haloperidol the tested drugs did not cause arrhythmias typically seen with the highly selective I(Kr)/hERG blocking drug dofetilide. For six of the eight drugs tested the effects on the embryonic rat heart were only seen at free drug concentrations that were much greater than those likely to occur in pregnant women taking antipsychotic medication. However, the safety margins for haloperidol and quetiapine were lower.

A microfabricated graphitic carbon column for high performance liquid chromatography.

We report the first development of a novel, planar, microfluidic, graphitic carbon separations column utilizing an array of graphitic micropillars of diamond cross-section as the chromatographic stationary phase. 795 nm femtosecond laser ablation was employed to subtractively machine fluidic architectures and a micropillared array in a planar, graphitic substrate as a monolithic structure. A sample injector was integrated on-chip, together with fluid-flow distribution architectures to minimize band-broadening and ensure sample equi-distribution across the micro-pillared column width. The separations chip was interfaced directly to the ESI probe of a Thermofisher Surveyor mass spectrometer, enabling the detection of test-mixture analytes following their differential retention on the micro-pillared graphitic column, thus demonstrating the exciting potential of this novel separations format. Importantly, unlike porous, graphitic microspheres, the temperature and pressure resilience of the microfluidic device potentially enables use in subcritical H(2)O chromatography.

Genotoxicity studies of a desealant solvent mixture, SR-51.

The Royal Australian Air Force (RAAF) has reported that personnel involved in F-111 fuel tank maintenance were concerned that exposure to a range of chemicals during the period 1977 to mid-1990s was the cause of health problems, including cancer. Particular concern was directed at SR-51, a desealant chemical mixture containing the following four solvents: aromatic 150 solvent (Aro150), dimethylacetamide, thiophenol (TP), and triethylphosphate. The present study examined the mutagenic potential of SR-51 using a range of well-known mutagen and genotoxin assays. The tests used were i) a modified version of the Ames test, ii) the mouse lymphoma assay, iii) the comet assay (a single-cell gel electrophoresis assay), and iv) a mouse micronucleus test. The modified Ames test used mixed bacterial strains in liquid suspension media. The Ames test results showed that SR-51 (tested up to the cytotoxic concentration of 36 microg/ml, 30 min incubation) in the presence and absence of S9 metabolic activation was not mutagenic. The mouse lymphoma assay used cultured mouse lymphoma cells in a microwell suspension method. The mouse lymphoma assay was also negative with SR-51 (tested up to the cytotoxic concentration of 22.5 microg/ml, 3 h incubation) in the presence and absence of S9 metabolic activation. The Comet assay, using cultured mouse lymphoma cells, showed no evidence of DNA damage in cells exposed up to the cytotoxic concentration of SR-51 at 11.25 microg/ml. The in-vivo mouse micronucleus test was undertaken in wild-type C57Bl6J male mice dosed orally with SR-51for 14 days with a single daily dose up to 360 mg/kg/day (the maximum-tolerated dose). No increases were observed in micronuclei (MN) frequency in bone marrow collected (24 h after final dose) from SR-51-treated mice compared to the number of MN observed in bone marrow collected from untreated mice. Tissues collected from treated mice at necropsy demonstrated a significant increase in spleen weights in the high dose mice. Gas chromatography analysis of SR-51 identified more than 40 individual components and an oxidation product, diphenyldisulfide derived from TP under conditions of mild heating. In conclusion, there was no evidence that SR-51 is mutagenic.

Effects of EDTA on the hydration mechanism of mineral trioxide aggregate.

Ethylenediaminetetraacetic acid (EDTA) is commonly used during the preparation of obstructed root canals that face a high risk of root perforation. Such perforations may be repaired with mineral trioxide aggregate (MTA). Due to EDTA's ability to chelate calcium ions, we hypothesized that EDTA may disrupt the hydration of MTA. Using scanning electron microscopy and energy-dispersive x-ray spectroscopy, we found that MTA specimens stored in an EDTA solution had no crystalline structure and a Ca/Si molar ratio considerably lower than those obtained for specimens stored in distilled water and normal saline. Poor cell adhesion in EDTA-treated MTA was also noted. X-ray diffraction indicated that the peak corresponding to portlandite, which is normally present in hydrated MTA, was not shown in the EDTA group. The microhardness of EDTA-treated specimens was also significantly reduced (p < 0.0001). These findings suggest that EDTA interferes with the hydration of MTA, resulting in decreased hardness and poor biocompatibility.

Dentin-pulp complex responses to carious lesions.

To understand the molecular events underlying the dentin-pulp complex responses to carious progression, we systematically analyzed tissue morphology and dentin matrix protein distribution in non-carious teeth and in teeth with enamel and dentin caries. Dentin matrix proteins analyzed included collagen type I, phosphophoryn (PP) and dentin sialoprotein (DSP), all of which play decisive roles in the dentin mineralization process. Human non-carious and carious third molar teeth were freshly collected, demineralized, and processed for hematoxylin and eosin staining. The ABC-peroxidase method was used for immunohistochemical staining of collagen type I, PP and DSP proteins using specific antibodies. In situ hybridization was also performed. In contrast to elongated odontoblasts in non-carious teeth, odontoblasts subjacent to dentin caries were cuboidal and fewer in number. The predentin zone was also dramatically reduced in teeth with dentin caries. The staining intensity for collagen type I, PP and DSP in the dentin-pulp complex increased progressively from non-carious teeth, to teeth with enamel and dentin caries. In situ hybridization studies showed DSP-PP mRNA expression in odontoblasts and dental pulp that was consistent with our immunohistochemical results. These results suggest that carious lesions stimulate the dentin-pulp complex to actively synthesize collagen type I, PP and DSP proteins. This response to carious lesions is likely to provide a basis for reparative and/or reactionary dentin formation.

Immunohistochemical characterization of rapid dentin formation induced by enamel matrix derivative.

The purpose of this study was to examine the pulpal expression of dentin-related proteins during enamel matrix derivative (EMD)-induced reparative dentin formation in a pulpotomy model in pig incisors. Pulpotomies were performed on 72 lower incisors in 24 adult miniature swine. The exposed pulp tissue was treated with EMD or covered with a calcium hydroxide paste (Dycal). At predefined time-points, ranging from 4 days to 12 weeks, experimental teeth were extracted and examined by use of light microscopy, and expression of dentin-related proteins in the pulps was investigated by immunohistochemistry, using antibodies against type I collagen, dentin sialoprotein (DSP), sheathlin, and EMD. In all EMD-treated teeth a substantial amount of reparative dentin formation was observed. The amount of reparative dentin in calcium hydroxide-treated teeth was significantly smaller than in EMD-treated teeth (P < 0.005) and was less effective in bridging the pulpal wounds. Immunohistochemistry demonstrated that enamel matrix proteins were present in detectable amounts at the application site for about 4 weeks. Moreover, the expression of proteins related to dentin formation in the wounded pulp tissue was about 2 weeks advanced in EMD-treated teeth. These findings demonstrate that enamel matrix molecules have the capacity to induce rapid pulpal wound healing in pulpotomized teeth, and suggest that the longevity and continued presence of enamel matrix macromolecules at the application site can be utilized to stimulate growth and repair of dentin over a period consistent with a favorable clinical outcome.

Effects of dexamethasone, vitamin A and vitamin D3 on DSP-PP mRNA expression in rat tooth organ culture.

Vitamin A, 1,25-dihydroxyvitamin D3 and dexamethasone are well-characterized hydrophobic molecules whose biological actions are mediated via different members of the nuclear hormone receptor family. We report here their actions on tooth formation at the molecular level. We have tested the effects of these compounds on osteopontin (OPN), dentin sialoprotein (DSP-PP), and collagen type I expression in pre-mineralization and mineralization stage rat tooth organ cultures which mirror in vivo developmental patterns. These proteins are all believed to participate in the mineralization of dentin. 1,25-Dihydroxyvitamin D3 up-regulated OPN, but had no effect on DSP-PP mRNA expression. Vitamin A up-regulated DSP-PP expression as did dexamethasone. Dexamethasone also up-regulated collagen type I expression. Our results suggest that 1,25-dihydroxyvitamin D3 does not modulate dentin mineralization by directly affecting DSP-PP expression. Vitamin A likely contributes to dentin mineralization by up-regulating DSP-PP expression. Finally, the up-regulation of DSP-PP expression in tooth germ cultures treated with dexamethasone suggests that its application to patient's dental pulp might promote increased extracellular matrix synthesis and mineralization in the pulp and may explain the narrowing of the dental pulp cavity in patients undergoing long-term dexamethasone administration.

Replacement of ether with alternate volatile anesthetics for collection of rat serum used in embryo culture.

BACKGROUND: The traditional anesthetic used for collection of the serum culture medium for whole rat embryo culture studies has been ether. However ethical concerns have been raised due to the irritant nature of the vapour and safety concerns due to the risk of fire. METHODS: Growth and development of gestation day 9.5 rat embryos cultured for 48 h in serum collected from rats anesthetised with either ether, isoflurane or halothane were compared. RESULTS: There were no differences in any of the parameters used to assess embryonic development when embryos were grown in serum collected using either ether or isoflurane anesthetics. However, when embryos grown in serum collected using ether or halothane were compared, embryonic development was similar in all respects, except for a reduced number of embryos turned to become fully dorsally convex in the halothane group (p <0.05). CONCLUSIONS: The data indicate that isoflurane is an appropriate alternative to ether for collection of the serum culture medium for whole rat embryo culture, while halothane may cause some delay of embryonic development.

Development of an odontoblast in vitro model to study dentin mineralization.

The aim of the present work was to characterize the odontoblastic proliferation, differentiation, and matrix mineralization in culture of the recently established M2H4 rat cell line. Proliferation was assessed by cell counts, differentiation by RT-PCR analysis, and mineralization by alizarin red staining, atomic absorption spectrometry, and FTIR microspectroscopy. The results showed that M2H4 cell behavior closely mimics in vivo odontoblast differentiation, with, in particular, temporally regulated expression of DMP-1 and DSPP. Moreover, the mineral phase formed by M2H4 cells was similar to that in dentin from rat incisors. Finally, because in mice, transforming growth factor (TGF)-beta1 over-expression in vivo leads to an hypomineralization similar to that observed in dentinogenesis imperfecta type II, effects of TGF-beta1 on mineralization in M2H4 cell culture were studied. Treatment with TGF-beta1 dramatically reduced mineralization, whereas positive control treatment with bone morphogenetic protein-4 enhanced it, suggesting that M2H4 cell line is a promising tool to explore the mineralization mechanisms in physiopathologic conditions.

The conservation and regulation of rat DSP-PP gene.

Two highly expressed noncollagenous proteins associated with dentin mineralization, dentin sialoprotein (DSP) and phosphophoryn (PP), are encoded by a single DSP-PP transcript. To better understand how DSP-PP transcripts are regulated, we have determined the DSP-PP transcription start site, sequenced its 5' flanking region, and analyzed the transcriptional activity of the gene promoter out to -1615 bp. Comparison of the rat cDNA sequence with the mouse, rat and human genes clearly indicates high sequence conservation within the DSP-PP 5' flanking region, implicating the possible presence of highly conserved gene regulatory cis elements. Among a number of conserved transcription sites identified in the 5' flanking region, we demonstrate that the conserved Y box sequence (ATTGG) can specifically bind nuclear extracts from mouse MDPC23 cells. This sequence (located within the -57 bp/-52 bp 5' flanking region) therefore likely represents one DSP-PP transcriptional regulatory sequence.

Phosphate and calcium uptake by rat odontoblast-like MRPC-1 cells concomitant with mineralization.

It has been suggested that odontoblasts are instrumental in translocating Ca2+ and inorganic phosphate (Pi) ions during the mineralization of dentin. The aim of this study was to characterize cellular Pi and Ca2+ uptake in the novel rat odontoblast-like cell line mineralizing rat pulpal cell line (MRPC) 1 during mineralization to see if changes in the ion transport activity would occur as the cultures develop and begin forming a mineralized matrix. MRPC-1 cells were cultured in chemically defined medium containing ascorbate and Pi, and cultures were specifically analyzed for cellular P, and Ca2+ uptake activities and expression of type II high-capacity Na+-Pi cotransporters. The odontoblast-like phenotype of the cell line was ascertained by monitoring the expression of collagen type I and dentin phosphopoprotein (DPP). Mineralized nodule formation started at day 9 after confluency and then rapidly increased. Ca2+ uptake by the cells showed a maximum during the end of the proliferative phase (days 5-7). Pi uptake declined to a basal level during proliferation and then was up-regulated simultaneously with the onset of mineralization to a level fourfold of the basal uptake, suggesting an initiating and regulatory role for cellular Pi uptake in mineral formation. This up-regulation coincided with a conspicuously increased glycosylation of NaPi-2a, indicating an activation of this Na+-Pi cotransporter. The study showed that MRPC-1 cells express an odontoblast-like phenotype already at the onset of culture, but that to mineralize the collagenous extracellular matrix (ECM) that formed, a further differentiation involving their ion transporters is necessary.

Testicular changes induced by chronic exposure to the herbicide formulation, Tordon 75D (2,4-dichlorophenoxyacetic acid and picloram) in rats.

The second most used herbicide in the Vietnam war was Agent White, which contained the active components 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-amino-3,5,6-trichloropicolinic acid (picloram). The herbicide formulation Tordon 75D is similar in terms of its active components to Agent White and is currently used by the agricultural industry in Australia. As part of an investigation into the possible adverse effects of this herbicide on male reproductive performance, groups of five male rats were gavaged 5 days a week for 9 weeks with either 0.125 ml/kg (low dose), 0.25 ml/kg (middle dose), or 0.5 ml/kg (high dose) Tordon 75D or water (controls). The high dose corresponded to 150 mg/kg body weight 2,4-D and 37.5 mg/kg picloram acid equivalents. At the end of the treatment period, the testes were collected, weighed, and examined histologically and blood samples were taken to determine serum testosterone. Groups of high dose animals were also examined after 1, 2, and 4 weeks treatment. The 9 weeks treatment with Tordon 75D caused severe reduction in testicular weight in some high dose animals. Histologically, the small testes showed shrunken tubules with germ cell depletion. This damage was still evident in some rats following a 21 weeks recovery period suggesting that the testicular damage was permanent. Testicular damage was not due to endocrine disruption as there were no significant differences in the serum concentration of testosterone in control animals compared to Tordon 75D-treated animals. Blood levels associated with the high dose were determined in a separate study and were much higher than those likely to be obtained by occupational exposure to this herbicide.

A study of the potential for a herbicide formulation containing 2,4-d and picloram to cause male-mediated developmental toxicity in rats.

Male Vietnam veterans have repeatedly expressed concern that exposure to herbicides in Vietnam may have caused birth defects in their offspring. The second most used herbicide was a mixture of 2,4-D and picloram called Agent White. This study is an investigation into the possible male-mediated reproductive toxicology of this herbicide. Male rats were gavaged for 5 days per week for 9 weeks with a mixture of 2,4-D and picloram called Tordon 75D(R) (the Australian derivative of Agent White). Three doses were tested; the high dose was considered the maximum tolerated dose. Each male was mated with two untreated females during weeks 2 and 3, 4 and 5, and 8 and 9 of treatment, and with four untreated females after an 11-week recovery period. Negative controls were males dosed with distilled water, and positive controls were males dosed with cyclophosphamide at 5.1 mg/kg/day. All mated females were killed on day 20 of gestation, and the fetuses were weighed and examined for either structural malformations or skeletal development. Litter size, fetal weight, and malformation rate were all unaffected by treatment. The cyclophosphamide positive controls showed the expected large increase in postimplantation loss. In general, within the limitations of the power of the study, the results did not show any evidence that exposure to a herbicide formulation containing 2,4-D and picloram is likely to cause male-mediated birth defects or other adverse reproductive outcomes.

PM(10)-exposed macrophages stimulate a proinflammatory response in lung epithelial cells via TNF-alpha.

There is now considerable evidence for an association between the levels of particulate air pollution [particulate matter <10 microm in aerodynamic diameter (PM(10))] and various adverse health endpoints. The release of proinflammatory mediators from PM(10)-exposed macrophages may be important in stimulating cytokine release from lung epithelial cells, thus amplifying the inflammatory response. A549 cells were treated with conditioned media from monocyte-derived macrophages stimulated with PM(10), titanium dioxide (TiO(2)), or ultrafine TiO(2). We demonstrate that only conditioned media from PM(10)-stimulated macrophages significantly increased nuclear factor-kappaB and activator protein-1 DNA binding, enhanced interleukin-8 (IL-8) mRNA levels as assessed by RT-PCR, and augmented IL-8 protein levels, over untreated controls. Furthermore, PM(10)-conditioned media also caused transactivation of IL-8 as determined by an IL-8-chloramphenicol acetyl transferase reporter. Analysis of these conditioned media revealed marked increases in tumor necrosis factor-alpha (TNF-alpha) and protein levels and enhanced chemotactic activity for neutrophils. Preincubation of conditioned media with TNF-alpha-neutralizing antibodies significantly reduced IL-8 production. These data suggest that PM(10)-activated macrophages may amplify the inflammatory response by enhancing IL-8 release from lung epithelial cells, in part, via elaboration of TNF-alpha.