Fibroblast growth factor-2 over-rides insulin-like growth factor-I induced proliferation and cell survival in human neuroblastoma cells

The insulin-like growth factor (IGF) system is a key regulator of cell growth, survival and differentiation, and these functions are co-modulated by other growth factors including fibroblast growth factor-2 (FGF-2). To investigate IGF/FGF interactions in neuronal cells, we employed neuroblastoma cells (SK-N-MC). In serum free conditions proliferation of the SK-N-MC cells was promoted by IGF-I (25 ng/ml), but blunted by FGF-2 (50 ng/ml). IGF-I-induced proliferation was abolished in the presence of FGF-2 even when IGF-I was used at 100 ng/ml. In addition to our previously described FGF-2 induced proteolytic cleavage of IGFBP-2, we found that FGF-2 increased IGFBP-6 levels in conditioned medium (CM) without affecting IGFBP-6 mRNA abundance. Modulation of IGFBP-2 and -6 levels were not significant mechanisms involved in the blockade of IGF-I action since the potent IGF-I analogues [QAYL]IGF-I and des(1-3)IGF-I (minimal IGFBP affinity) were unable to overcome FGF-2 inhibition of cell proliferation. FGF-2 treated cells showed morphological differentiation expressing the TUJ1 neuronal marker while cells treated with IGF-I alone showed no morphological change. When IGF-I was combined with FGF-2, however, cell morphology was indistinguishable from that seen with FGF-2 alone. FGF-2 inhibited proliferation and enhanced differentiation was also associated with a 70% increase in cell death. Although IGF-I alone was potently anti-apoptotic (60% decreased), IGF-I was unable to prevent apoptosis when administrated in combination with FGF-2. Gene-array analysis confirmed FGF-2 activation of the intrinsic and extrinsic apoptotic pathways and blockade of IGF anti-apoptotic signaling. FGF-2, directly and indirectly, overcomes the proliferative and anti-apoptotic activity of IGF-I by complex mechanisms, including enhancement of differentiation and apoptotic pathways, and inhibition of IGF-I induced anti-apoptotic signalling. Modulation of IGF binding protein abundance by FGF-2 does not play a significant role in inhibition of IGF-I induced mitogenesis.

Mating system variation in the hermaphroditic brooding coral, Seriatopora hystrix

Self-compatible, hermaphroditic marine invertebrates have the potential to self-fertilize in the absence of mates or under sperm-limited conditions, and outcross when sperm is available from a variety of males. Hence, many hermaphroditic marine invertebrates may have evolved mixed-mating systems that involve facultative self-fertilization. Such mixed-mating strategies are well documented for plants but have rarely been investigated in animals. Here, I use allozyme markers to make estimates of selfing from population surveys of reef slope and reef flat sites, and contrast this with direct estimates of selfing from progeny-array analysis, for the brooding coral Seriatopora hystrix. Consistent heterozygote deficits previously reported for S. hystrix suggests that inbreeding (including the extreme of selfing) may be common in this species. I detected significant levels of inbreeding within populations (FIS=0.48) and small but significant differentiation among all sites (FST=0.04). I detected no significant differentiation among habitats (FHT=0.009) though among site differentiation did occur within the reef slope habitat (FSH=0.06), but not within the reef flat habitat (FSH=0.015). My direct estimates of outcrossing for six colonies and their progeny from a single reef flat site revealed an intermediate value (tm (±s.d.)=0.53±0.20). Inbreeding coefficients calculated from progeny arrays (Fe=0.31) were similar to indirect estimates based on adult genotype frequencies for that site (FIS=0.38). This study confirms that the mating system of this brooding coral is potentially variable, with both outcrossing and selfing.

Lipids and FA analysis of canine prostate tissue

It is widely reported that an association exists between dietary fat intake and the incidence of prostate cancer in humans. To study this association, there is a need for an animal model where prostate carcinogenesis occurs spontaneously. The canine prostate is considered a suitable experimental model for prostate cancer in humans since it is morphologically similar to the human prostate and both humans and dogs have a predisposition to benign and malignant prostate disease. In this study, the FA and lipids profiles of the normal canine prostate tissue from nine dogs were examined. The total lipid content of the canine prostate tissue was 1.7±0.5% (wet weight). The lipid composition analysis using TLC-FID showed that the two major lipid classes were phospholipids and TAG. Total FA, phospholipid, and TAG FA analysis showed that the major FA were palmitic acid (16∶0), stearic acid (18∶0), oleic acid (18∶1), linoleic acid (18∶2n−6), and arachidonic acid (20∶4n−6), The n−3 FA were present at <3% of total FA and included α-linolenic acid (18∶3n−3) (in total and TAG tissue FA), EPA (20∶5n−3) (not in TAG), and DHA (22∶6n−3) (not in TAG). The n−3/n−6 ratio was 1∶11, 1∶13, and 1∶8 in total, phospholipid, and TAG FA, respectively. This study shows the canine prostate has a low level of n−3 FA and a low n−3/n−6 ratio. This is perhaps due to low n−3 content of the diet of the dogs. FA analysis of dogfoods available in Australia showed that the n−3 content in both supermarket and premium bran dogfoods was <3% (wet weight), and the n−3/n−6 ratio was low.

Identification of surface heterogeneity effects in cyclic voltammograms derived from analysis of an individually addressable gold array electrode

Voltammetric behavior at gold electrodes in aqueous media is known to be strongly dependent on electrode polishing and history. In this study, an electrode array consisting of 100 nominally identical and individually addressable gold disks electrodes, each with a radius of 127 µm, has been fabricated. The ability to analyze both individual electrode and total array performance enables microscopic aspects of the overall voltammetric response arising from variable levels of inhomogeneity in each electrode to be identified. The array configuration was initially employed with the reversible and hence relatively surface insensitive [Ru(NH₃)₆]^3+/2+ reaction and then with the more highly surface sensitive quasi-reversible [Fe(CN)₆]^3−/4− process. In both these cases, the reactants and products are solution soluble and, at a scan rate of 50 mV s⁻¹, each electrode in the array is assumed to behave independently, since no evidence of overlapping of the diffusion layers was detected. As would be expected, the variability of the individual electrodesʼ responses was significantly larger than found for the summed electrode behavior. In the case of cytochrome c voltammetry at a 4,4′-dipyridyl disulfide modified electrode, a far greater dependence on electrode history and electrode heterogeneity was detected. In this case, voltammograms derived from individual electrodes in the gold array electrode exhibit shape variations ranging from peak to sigmoidal. However, again the total response was always found to be well-defined. This voltammetry is consistent with a microscopic model of heterogeneity where some parts of each chemically modified electrode surface are electroactive while other parts are less active. The findings are consistent with the common existence of electrode heterogeneity in cyclic voltammetric responses at gold electrodes, that are normally difficult to detect, but fundamentally important, as electrode nonuniformity can give rise to subtle forms of kinetic and other forms of dispersion.

Comparative analysis of microRNA expression in mouse and human brown adipose tissue

BACKGROUND: In small mammals brown adipose tissue (BAT) plays a predominant role in regulating energy expenditure (EE) via adaptive thermogenesis. New-born babies require BAT to control their body temperature, however its relevance in adults has been questioned. Active BAT has recently been observed in adult humans, albeit in much lower relative quantities than small mammals. Comparing and contrasting the molecular mechanisms controlling BAT growth and development in mice and humans will increase our understanding or how human BAT is developed and may identify potential therapeutic targets to increase EE. MicroRNAs are molecular mechanisms involved in mouse BAT development however, little is known about the miRNA profile in human BAT. The aims of this study were to establish a mouse BAT-enriched miRNA profile and compare this with miRNAs measured in human BAT. To achieve this we firstly established a mouse BAT enriched-miRNA profile by comparing miRNAs expressed in mouse BAT, white adipose tissue and skeletal muscle. Following this the BAT-enriched miRNAs predicted to target genes potentially involved in growth and development were identified.

METHODS: MiRNA levels were measured using PCR-based miRNA arrays. Results were analysed using ExpressionSuite software with the global mean expression value of all expressed miRNAs in a givensample used as the normalisation factor. Bio-informatic analyses was used to predict gene targets followed by Ingenuity Pathway Analysis.

RESULTS: We identified 35 mouse BAT-enriched miRNAs that were predicted to target genes potentially involved in growth and development. We also identified 145 miRNAs expressed in both mouse and human BAT, of which 25 were enriched in mouse BAT. Of these 25 miRNAs, miR-20a was predicted to target MYF5 and PPARγ, two important genes involved in brown adipogenesis, as well as BMP2 and BMPR2, genes involved in white adipogenesis. For the first time, 69 miRNAs were identified in human BAT but absent in mouse BAT, and 181 miRNAs were expressed in mouse but not in human BAT.

CONCLUSION: The present study has identified a small sub-set of miRNAs common to both mouse and human BAT. From this sub-set bioinformatics analysis suggested a potential role of miR-20a in the control of cell fate and this warrants further investigation. The large number of miRNAs found only in mouse BAT or only in human BAT highlights the differing molecular profile between species that is likely to influence the functional role of BAT across species. Nevertheless the BAT-enriched miRNA profiles established in the present study suggest targets to investigate in the control BAT development and EE.

A comparison of collection techniques for gene expression analysis of human oral taste tissue

Variability in human taste perception is associated with both genetic and environmental factors. The influence of taste receptor expression on this variability is unknown, in part, due to the difficulty in obtaining human oral tissue that enables quantitative expression measures of taste genes. In a comparison of six current techniques (Oragene RNeasy Kit, Isohelix swab, Livibrush cytobrush, tongue saliva, cheek saliva collection, and fungiform papillae biopsy), we identify the fungiform papillae biopsy is the optimal sampling technique to analyse human taste gene expression. The fungiform papillae biopsy resulted in the highest RNA integrity, enabling amplification of all the assessed taste receptor genes (TAS1R1, TAS1R2, TAS1R3, SCNN1A and CD36) and taste tissue marker genes (NCAM1, GNAT3 and PLCβ2). Furthermore, quantitative expression was observed in a subset of taste genes assessed from the saliva collection techniques (cheek saliva, tongue saliva and Oragene RNA kit). These saliva collection techniques may be useful as a non-invasive alternative sampling technique to the fungiform papillae biopsy. Identification of the fungiform papillae biopsy as the optimal collection method will facilitate further research into understanding the effect of gene expression on variability in human taste perception.

Functional analysis of natriuretic peptide receptors in the bladder of the toad, bufo marinus

This study aimed to localize and characterize natriuretic peptide binding sites in the urinary bladder of Bufo marinus and to then examine the effect of natriuretic peptides on the bladder vascular tone and water reabsorption in isolated perfused bladder preparations. Specific ¹²⁵I-rat atrial natriuretic peptide (¹²⁵I-rANP) binding sites were present on blood vessels, muscle, and epithelium. In tissue sections and/or isolated membranes, the binding was completely displaced by frog ANP, rat ANP, and porcine C-type natriuretic peptide (CNP; membranes only). However, a reduction in binding was observed after incubation with ¹²⁵I-rANP and 1 μM of the natriuretic peptide receptor-C (NPR-C) ligand C-ANF, but residual binding remained suggesting the presence of two distinct binding sites. Electrophoresis of bladder membranes cross-linked to ¹²⁵I-rANP identified two bands at approximately 70 and 140 kDa that correspond to the monomeric mass of NPR-C and the guanylate cyclase receptors, respectively. Furthermore, the presence of natriuretic peptide receptor-A and NPR-C mRNA in the bladder was demonstrated with reverse transcription–polymerase chain reaction. In addition, rat ANP, frog ANP, and porcine CNP stimulated a significant increase in cGMP generation in bladder membrane preparations, which indicated the presence of guanylate cyclase-linked receptors. In perfused bladder preparations, arginine vasotocin increased perfusion pressure and water permeability. The infusion of frog ANP or porcine CNP failed to alter perfusion pressure or water reabsorption in the presence or absence of arginine vasotocin. This study identified a well-developed natriuretic peptide receptor system in the urinary bladder of B. marinus but the function of the receptors remains unclear.

Fighting a tobacco rollback : a political analysis of the 1994 contraband crisis in Canada

We identify factors that led a regional government (Quebec, Canada) to opt for a reduction of its tobacco tax to combat tobacco smuggling. Then we explore the fallout of Quebec's tobacco-tax rollback on its tobacco control policy. We conducted qualitative research using a case-study design and multiple sources of data. We applied the Advocacy Coalition Framework in respect of data collection and analysis. Advocates of the tobacco-tax rollback framed the contraband problem in a way that won the support of an array of actors. However, anti-tobacco activists succeeded in convincing the government to invest more in tobacco control. The new resources were instrumental in enhancing the activists' ability to promote legislative measures. Our approach sheds light on the tobacco industry's strategy to have governments reducing their tobacco tax. Quebec offers an example of how tobacco control activists can transform defeat into the cornerstone of a comprehensive tobacco control policy.

Titanium–nickel shape memory alloy foams for bone tissue engineering

Titanium–nickel (TiNi) shape memory alloy (SMA) foams with an open-cell porous structure were fabricated by space-holder sintering process and characterized by scanning electron microscopy (SEM) and X-ray diffraction (XRD) analysis. The mechanical properties and shape memory properties of the TiNi foam samples were investigated using compressive test. Results indicate that the plateau stresses and elastic moduli of the foams under compression decrease with the increase of their porosities. The plateau stresses and elastic moduli are measured to be from 1.9 to 38.3 MPa and from 30 to 860 MPa for the TiNi foam samples with porosities ranged from 71% to 87%, respectively. The mechanical properties of the TiNi alloy foams can be tailored to match those of bone. The TiNi alloy foams exhibit shape memory effect (SME), and it is found that the recoverable strain due to SME decreases with the increase of foam porosity.

Process analysis of the solution strategies used for problems contained in the Minnesota Paper Board Form Board test

This thesis reviews progress toward an understanding of the processes involved in the solution of spatial problems. Previous work employing factor analysis and information processing analysis is reviewed and the emphasis on variations in speed and accuracy as the major contributers to individual differences is noted. It is argued that the strategy used by individuals is a preferable explanatory concept for identifying the cognitive substratum necessary for problem solving. Using the protocols obtained from subjects solving The Minnesota Paper Form Board (Revised), a test commonly regarded as measuring skill in spatial visualization, a number of different strategies are isolated. Assumptions as to the task variants which undergird these strategies are made and tested experimentally. The results suggest that task variants such as the size of the stimulus and the shape of the pieces interact with subject variables to produce the operating strategy. Skill in problem solving is revealed in the ability to structure the array, to hold a structured image and to reduce the number of answers requiring intensive processing. The interaction between task and subject variables results in appropriate or inappropriate strategies which in turn affect speed and accuracy. Results suggest that strategy formation and usage are the keys to explaining individual differences and an heuristic model is presented to explain the performance of individual subjects on the problems involved in the Minnesota Paper Form Board. The model can be used to predict performance on other tests; and as an aid to teaching subjects experiencing difficulties. The model presented incorporates strategy variation and is consequently mores complex than previously suggested models. It is argued that such complexity is necessary to explain the nature of a subject's performance and is also necessary to perform diagnostic evaluation. Certain structural -features of the Minnesota Paper Form Board are questioned and suggestions for improvement included. The essential explanatory function of the strategy in use makes the prevalent group administration approach suspect in the prediction of future performance in spatial or vocational activity.

Steroidogenesis in cultured mammalian glial cells

A protocol for culturing mammalian type 1 astrocytic cells, using female post-natal rat cerebral cortical tissue, was established and refined for use in steroidogenic metabolic studies incorporating progestin radioisotopes. Cultures were characterised for homogeneity using standard morphological and immunostaining techniques. Qualitative and quantitative studies were conducted to characterise the progesterone (P) metabolic pathways present in astrocytes in vitro. Of particular interest was the formation of the P metabolite, 5á-pregnan-3á-ol-20-one (THP). THP is a GABA(A) receptor agonist, believed to play a vital role in neural functioning and CNS homeostasis. One aim of this study was to observe any modulatory effects selected neuroactive ligands have on the conversion of P into THP, in an attempt to link astrocytic steroidogenesis with neuronal control. In qualitative studies, chromatographic procedures were used to establish the progestin profile of cerebral cortical astrocytes. Tritiated P, DHP (5á-pregnan-3,20-dione) and THP incurbates were preliminary fractionated by either normal phase (NP) or reverse phase (RP) high performance liquid chromatography (HPLC). The radiometabolites associated with each fraction were further chromatographed, before and/or after chemical derivatistation, by the aforemention HPLC procedures and thin layer chromatography (TLC). Steroid radiometabolites were tentatively identified by comparing their chromatographic mobility with authentic steroids. The identity of the main putative 5á-reduced P metabolities, DHP, THP and 5á-pregnan-3á,20á-diol (20áOH-THP) were further confirmed by isotopic dilution analysis. Their conclusive identification, along with the tentative identification of 20á-hydroxypreg-4-en-3-one (20áOH-P) and 20á-hydroxy-5á-pregnan-3-one (20áOH-DHP), verify the localisation of 5á-reductase, 3á-hydroxy steroif oxidoreductase (HSOR), and 20á-HSOR activity in the cultured astrocytes utilised in this study programme. Other minor metabolites detected were tentatively identified, including 5á-pregnan-3á,21-diol-20-one (THDoc), indicating the presence of 21-hydroxylase enzymatic activity. THDoc, like THP, is a GABA(A) receptor agonist. The chemical and physical characterisation of several yet unidentified progestin metabolites, associated with a highly polar RP HPLC fraction (designated RP peak 1*), indicate the presence of one or more extra hydroxylase enzymes. Quantitative analysis included a preliminary study. In this study, the percentage yields of radiometabolites formed in cultures incubated with increasing substrate concentrations of (3)H-P for 24 hours were determined. At the lower concentrations examined (ie 0.5 to 50nM), the metabolites associated with the polar RP HPLC fraction (RP peak 1*) collectively have the highest percentage yield. They are subsequently considered metabolic end products of degradative catabolic P pathways. The percentage yield of THP peaks in the medium concentration ranges (ie 5 to 500nM), whereas DHP remains fairly static at a low level with increasing concentration. Both DHP and THP are considered metabolic pathway intermediates. The percentage yield of 20áOH-THP continues to increase with increasing concentration over 5nM, superseding THP approaching the highest concentration examined (5000nM). This indicated the formation of 20áOH-THP does not occur entirely via THP. 20áOH-THP also possibly serves as the direct intermediate in the formation of the main radiometabolites associated with RP peak 1*. A time/yield study incorporating incubation times from one to 24 hours was also conducted. The full array of radiometabolites (individually or in groups) formed in astrocyte cultures incubated with 50nM tritiated P, DHP of THP, were assayed. Cultures were observed to rapidly convert any DHP into THP, showing astrocytic 3á-HSOR activity is very high. The study also showed 5á-reduction (ie the conversation of P into DHP) is the rate limiting reaction in the two step conversion of P into THP. 5á-Reduction also appears to be a rate limiting step in the formation of 20á-hydroxylated metabolites in astrocytes. Cultures incubated with the tritiated 5á-reduced pregnanes from one to four hours form greater quantities to 20á-hydroxylated radiometabolites compared to cultures incubated with (3)H-P. The time yield/studies also provided further evidence the unidentified polar radiometabolites associated with RP peak 1* are metabolic end products. For the P and DHP incubates, the collective formation of the aforementioned polar radiometabolites initially lags behind the formation of THP. As the formation of the latter begins to plateau with increasing time between four to 24 hours, the net yield of radiometabolites associated with RP peak 1* continues to rise. The time/yield studies also indicate 5á-reduction and perhaps 3á-hydroxylation are pre-requisite steps in the formation of the polar metabolites. Cultures incubated with the 5á-reduced progestins from one to four hours form higher yields of the radiometabolites associated with RP peak 1* compared to cultures incubated with P as substrate. The net yields of the radiometabolites associated with RP peak 1* for cultures incubated with THP were substantially higher compared to cultures incubated with DHP after equivalent times. The effect selected neuroligands have on the yield of radiometabolites formed by cultured astrocytes incubated with 50nM (3)H-P was also examined. Dibutyryl cyclic adenosine monophosphate (DBcAMP), not actually a neuroligand per se, but an analog of the intracellular secondary messenger cAMP, was also utilised in these studies. The inhibitory neurotransmitter ă-amino-nbutyric acid (GABA), DBcAMP and isoproterenol (a â-adrenergic receptor agonist) all quickly induce a transient but substantial increase in 20á-HSOR activity in cultured astrocytes. Cultures pretreated with these three compounds (10, 20 and 1µM respectively) form substantially higher yields of 20á-hydroxylated metabolites, including 20áOH-THP (between 200 to 580% greater), when incubated with 50nM (3)H-P for one to four hours. These increases also coincide with increases in the net yield of metabolites formed (by 16 to 48%). The same pre-treated cultures form significantly lower yields of THP, by 25 to 41%, after one hour. This is most likely due to the increased metabolism of any formed THP into 20áOH-THP. Octopamine (an á-adrenergic agonist) only induces a slight increase in 20á-HSOR activity, having relatively little effect on the yield of 20áOH-THP formed. Pretreatment with octopamine induces a significant increase in the yield of THP for cultures incubated with (3)H-P for four hours (by 24%). The increase in THP formation appears to be due to an increase in 3á-HSOR activity, as judged by the concomitant drop in the yield of the 5á-reduced, 3-keto substrates. An increase in 5á-reductase activity cannot be excluded however. Isoproterenol appears to induce an increase in 5á-reductase activity as isoproterenol appears to induce an increase in 5á-reductase activity as isoproterenol one and four hour incubates have higher yields of DHP. This is in contrast to the other three incubates. After 12 hours, all incubates have higher yields of THP (15-30%).

Diagnosis of {alpha}+-thalassemias by determining the ratio of the two {alpha}-globin gene copies by oligonucleotide hybridization and melting curve analysis

Most cases of {alpha}-thalassemia result from large deletions at the {alpha}-globin locus (1). The {alpha}-globin gene cluster contains a tandem array of 2 nearly identical {alpha}-globin genes (HBA; Fig. 1A ) (2). The {alpha}0-thalassemias are characterized by deletions that inactivate both {alpha}-globin genes of a given chromosome, whereas in {alpha}+-thalassemias, one gene remains functional. The most widespread {alpha}+-thalassemias are those designated –{alpha}3.7 and –{alpha}4.2, according to the lengths of the deleted fragments (3).

Analysis of children's merging errors

Array