Increased expression of transglutaminase-1 and PPARgamma after vitamin E treatment in human keratinocytes

In skin, vitamin E acts as the predominant lipophilic antioxidant with a protective function against irradiation and oxidative stress. In addition to that, vitamin E can also modulate signal transduction and gene expression. To study whether the four natural tocopherol analogues (α-, β-, γ-, δ-tocopherol) can influence transcriptional activity by modulating the activity of nuclear receptors, a human keratinocytes cell line (NCTC 2544) was transfected with plasmids containing the luciferase reporter gene under control by direct repeat elements (DR1–DR4), representing binding sites for four different classes of nuclear receptors. In this model, the tocopherols positively modulated only the reporter construct containing a consensus element for peroxisome proliferator-activated receptors (PPARs). The induction was strongest with γ-tocopherol and was most likely the direct consequence of stimulation of PPARγ protein expression in keratinocytes. Vitamin E treatment also led to increased expression of a known PPARγ target gene involved in terminal keratinocytes differentiation, the transglutaminase-1.

Transport in the blood of an anti-tumor water-soluble rutheniun cyclopentadienyl complex: a fluorescence study on albumin binding

Dissertação de mestrado, Qualidade em Análises, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2014

The bacterial chromosome segregation protein Spo0J spreads along DNA from parS nucleation sites

Regulation of chromosome inheritance is essential to ensure proper transmission of genetic information. To accomplish accurate genome segregation, cells organize their chromosomes and actively separate them prior to cytokinesis. In Bacillus subtilis the Spo0J protein is required for accurate chromosome segregation and it regulates the developmental switch from vegetative growth to sporulation. Spo0J is a DNA-binding protein that recognizes at least eight identified parS sites located near the origin of replication. As judged by fluorescence microscopy, Spo0J forms discrete foci associated with the oriC region of the chromosome throughout the cell cycle. In an attempt to determine the mechanisms utilized by Spo0J to facilitate productive chromosome segregation, we have investigated the DNA binding activity of Spo0J. In vivo we find Spo0J associates with several kilobases of DNA flanking its specific binding sites (parS) through a parS-dependent nucleation event that promotes lateral spreading of Spo0J along the chromosome. Using purified components we find that Spo0J has the ability to coat non-specific DNA substrates. These 'Spo0J domains' provide large structures near oriC that could potentially demark, organize or localize the origin region of the chromosome.

Small molecule–biopolymer interactions : Ultraviolet–visible and fluorescence spectroscopy and chemometrics

Interactions between small molecules with biopolymers e.g. the bovine serum albumin (BSA protein), are important, and significant information is recorded in the UV–vis and fluorescence spectra of their reaction mixtures. The extraction of this information is difficult conventionally and principally because there is significant overlapping of the spectra of the three analytes in the mixture. The interaction of berberine chloride (BC) and the BSA protein provides an interesting example of such complex systems. UV–vis and fluorescence spectra of BC and BSA mixtures were investigated in pH 7.4 Tris–HCl buffer at 37 °C. Two sample series were measured by each technique: (1) [BSA] was kept constant and the [BC] was varied and (2) [BC] was kept constant and the [BSA] was varied. This produced four spectral data matrices, which were combined into one expanded spectral matrix. This was processed by the multivariate curve resolution–alternating least squares method (MCR–ALS). The results produced: (1) the extracted pure BC, BSA and the BC–BSA complex spectra from the measured heavily overlapping composite responses, (2) the concentration profiles of BC, BSA and the BC–BSA complex, which are difficult to obtain by conventional means, and (3) estimates of the number of binding sites of BC.

Spectrometric and voltammetric studies of the interaction between quercetin and bovine serum albumin using warfarin as site marker with the aid of chemometrics

The interaction of quercetin, which is a bioflavonoid, with bovine serum albumin (BSA) was investigated under pseudo-physiological conditions by the application of UV–vis spectrometry, spectrofluorimetry and cyclic voltammetry (CV). These studies indicated a cooperative interaction between the quercetin–BSA complex and warfarin, which produced a ternary complex, quercetin–BSA–warfarin. It was found that both quercetin and warfarin were located in site I. However, the spectra of these three components overlapped and the chemometrics method – multivariate curve resolution-alternating least squares (MCR-ALS) was applied to resolve the spectra. The resolved spectra of quercetin–BSA and warfarin agreed well with their measured spectra, and importantly, the spectrum of the quercetin–BSA–warfarin complex was extracted. These results allowed the rationalization of the behaviour of the overlapping spectra. At lower concentrations ([warfarin] < 1 × 10−5 mol L−1), most of the site marker reacted with the quercetin–BSA, but free warfarin was present at higher concentrations. Interestingly, the ratio between quercetin–BSA and warfarin was found to be 1:2, suggesting a quercetin–BSA–(warfarin)2 complex, and the estimated equilibrium constant was 1.4 × 1011 M−2. The results suggest that at low concentrations, warfarin binds at the high-affinity sites (HAS), while low-affinity binding sites (LAS) are occupied at higher concentrations.

Discovery and characterization of IGFBP-mediated endocytosis in the human retinal pigment epithelial cell line ARPE-19

Insulin-like growth factor binding proteins (IGFBPs) are prime regulators of IGF-action in numerous cell types including the retinal pigment epithelium (RPE). The RPE performs several functions essential for vision, including growth factor secretion and waste removal via a phagocytic process mediated in part by vitronectin (Vn). In the course of studying the effects of IGFBPs on IGF-mediated VEGF secretion and Vn-mediated phagocytosis in the RPE cell line ARPE-19, we have discovered that these cells avidly ingest synthetic microspheres (2.0 μm diameter) coated with IGFBPs. Given the novelty of this finding and the established role for endocytosis in mediating IGFBP actions in other cell types, we have explored the potential role of candidate cell surface receptors. Moreover, we have examined the role of key IGFBP structural motifs, by comparing responses to three members of the IGFBP family (IGFBP-3, IGFBP-4 and IGFBP-5) which display overlapping variations in primary structure and glycosylation status. Coating of microspheres (FluoSpheres®, sulfate modified polystyrene filled with a fluorophore) was conducted at 37 °C for 1 h using 20 μg/mL of test protein, followed by extensive washing. Binding of proteins was confirmed using a microBCA assay. The negative control consisted of microspheres treated with 0.1% bovine serum albumin (BSA), and all test samples were post-treated with BSA in an effort to coat any remaining free protein binding sites, which might otherwise encourage non-specific interactions with the cell surface. Serum-starved cultures of ARPE-19 cells were incubated with microspheres for 24 h, using a ratio of approximately 100 microspheres per cell. Uptake of microspheres was quantified using a fluorometer and was confirmed visually by confocal fluorescence microscopy. The ARPE-19 cells displayed little affinity for BSA-treated microspheres, but avidly ingested large quantities of those pre-treated with Vn (ANOVA; p < 0.001). Strong responses were also observed towards recombinant formulations of non-glycosylated IGFBP-3, glycosylated IGFBP-3 and glycosylated IGFBP-5 (all p < 0.001), while glycosylated IGFBP-4 induced a relatively minor response (p < 0.05). The response to IGFBP-3 was unaffected in the presence of excess soluble IGFBP-3, IGF-I or Vn. Likewise, soluble IGFBP-3 did not induce uptake of BSA-treated microspheres. Antibodies to either the transferrin receptor or type 1 IGF-receptor displayed slight inhibitory effects on responses to IGFBPs and Vn. Heparin abolished responses to Vn, IGFBP-5 and non-glycosylated IGFBP-3, but only partially inhibited the response to glycosylated IGFBP-3. Our results demonstrate for the first time IGFBP-mediated endocytosis in ARPE-19 cells and suggest roles for the IGFBP-heparin-binding domain and glycosylation status. These findings have important implications for understanding the mechanisms of IGFBP actions on the RPE, and in particular suggest a role for IGFBP-endocytosis.

The highs (B1H) and lows (B1L) of human heart B1-adrenoceptors (AR)

The 1AR has two binding sites which can be activated to cause cardiostimulation. The first, termed, 1HAR (high affinity site of 1AR) is activated by noradrenaline and adrenaline and is blocked by relatively low concentrations of β-blockers including carvedilol (Kaumann and Molenaar, 2008). The other, termed, 1LAR (low affinity site of 1AR) has lower affinity for noradrenaline and adrenaline and is activated by some β-blockers including CGP12177 and pindolol, at higher concentrations than those required to block the receptor (Kaumann and Molenaar, 2008). (-)-CGP12177 is a non-conventional partial agonist that causes modest and transient increases of contractile force in human atrial trabeculae (Kaumann and Molenaar, 2008). These effects are markedly increased and maintained by inhibition of phosphodiesterase PDE3. The stimulant effects of (-)-CGP12177 at human β1ARs was verified with recombinant receptors (Kaumann and Molenaar, 2008). However, in a recent report it was proposed that the positive inotropic effects of CGP12177 are mediated through 3ARs in human right atrium (Skeberdis et al 2008). This proposal was not consistent with the lack of blockade of (-)-CGP12177 inotropic effects or increases in L-type Ca2+ current (ICa-L ) by the β3AR blocker 1 μM LY748,337 (Christ et al, 2010). On the otherhand, (-)-CGP12177 increases in inotropic effects and ICa-L were blocked by (-)-bupranolol 1-10 μM (Christ et al, 2010). Chronic infusion of (-)-CGP 12177 (10 mg/Kg/24 hours) for four weeks in an aortic constriction mouse model of heart failure caused an increase in left ventricular wall thickness, fibrosis and inflammation-related left ventricular gene expression levels. Christ T et al (2010) Br J Pharmacol, In press Kaumann A and Molenaar P (2008) Pharmacol Ther 118, 303-336 Skeberdis VA et al (2008) J Clin Invest, 118, 3219-3227

Role of low affinity beta1-adrenergic receptors in normal and diseased hearts

ROLE OF LOW AFFINITY β1-ADRENERGIC RECEPTOR IN NORMAL AND DISEASED HEARTS Background: The β1-adrenergic receptor (AR) has at least two binding sites, 1HAR and 1LAR (high and low affinity site of the 1AR respectively) which cause cardiostimulation. Some β-blockers, for example (-)-pindolol and (-)-CGP 12177 can activate β1LAR at higher concentrations than those required to block β1HAR. While β1HAR can be blocked by all clinically used β-blockers, β1LAR is relatively resistant to blockade. Thus, chronic β1LAR activation may occur in the setting of β-blocker therapy, thereby mediating persistent βAR signaling. Thus, it is important to determine the potential significance of β1LAR in vivo, particularly in disease settings. Method and result: C57Bl/6 male mice were used. Chronic (4 weeks) β1LAR activation was achieved by treatment with (-)-CGP12177 via osmotic minipump. Cardiac function was assessed by echocardiography and catheterization. (-)-CGP12177 treatment in healthy mice increased heart rate and left ventricular (LV) contractility without detectable LV remodelling or hypertrophy. In mice subjected to an 8-week period of aorta banding, (-)-CGP12177 treatment given during 4-8 weeks led to a positive inotropic effect. (-)-CGP12177 treatment exacerbated LV remodelling indicated by a worsening of LV hypertrophy by ??% (estimated by weight, wall thickness, cardiomyocyte size) and interstitial/perivascular fibrosis (by histology). Importantly, (-)-CGP12177 treatment to aorta banded mice exacerbated cardiac expression of hypertrophic, fibrogenic and inflammatory genes (all p<0.05 vs. non-treated control with aorta banding).. Conclusion: β1LAR activation provides functional support to the heart, in both normal and diseased (pressure overload) settings. Sustained β1LAR activation in the diseased heart exacerbates LV remodelling and therefore may promote disease progression from compensatory hypertrophy to heart failure. Word count: 270

Contribution of transmembrane domain V amino acids to β1l-adrenoceptor activity and affinity

Introduction. There are two binding sites on the β1-adrenoceptor (AR), β1H and β1L corresponding to high and low affinity binding sites respectively, which can be activated to cause cardiostimulation. Some β-blockers that block β1AR and β2ARs can activate β1LARs at higher concentrations than those required to cause blockade. The β2AR does not form a corresponding low affinity binding site and therefore we postulated that heterologous amino acids are responsible for the formation of β1LAR. Aim. To investigate whether heterologous amino acids of transmembrane domain V (TMDV) of β1AR and β2ARs contribute to β1LAR. Methods. β1ARs, β2ARs and mutant β1ARs containing all (β1(β2TMDV)AR) or single amino acids of TMDV of the β2AR were prepared and stably expressed in Chinese Hamster Ovary cells. Concentration-effect curves for cyclicAMP accumulation were carried out for (-)-CGP12177 in the absence or presence of (-)-bupranolol. Results. The potencies (pEC50) of (-)-CGP12177 were β2AR (9.24 ± 0.14, n = 5), β1(V230I)AR (9.07 ± 0.07, n = 10), β1(β2TMDV)AR (8.86 ± 0.10, n = 15), β1(R222Q)AR (8.09 ± 0.29, n = 6), β1AR (8.00 ± 0.11, n = 11). The affinities (pKB) of (-)-bupranolol were β2AR (9.82 ± 0.52, n = 5), β1(V230I)AR (7.64 ± 0.12, n = 8), β1(β2TMV)AR (8.06 ± 0.17, n = 8), β1(R222Q)AR (7.33 ± 0.23, n = 5), β1AR (7.23 ± 0.23, n = 5). Discussion. The potency of (-)-CGP12177 was higher at β2AR than at β1AR consistent with activation through a low affinity site at the β1AR (β1LAR). The presence of V230 in β1AR accounted for the lower potency of (-)-CGP 12177. The affinity of (-)-bupranolol was lower at β1AR compared to β2AR. The presence of V230 in β1AR accounted in part for the lower affinity. In conclusion TMDV of the β1AR contributes in part to the low affinity binding site of β1AR.

Contribution of transmembrane domain V amino acids to β1l-adrenoceptor activity and affinity

There are two binding sites on the β1-adrenoceptor (AR), β1H and β1L corresponding to high and low affinity binding sites respectively, which can be activated to cause cardiostimulation (reviewed Kaumann and Molenaar, 2008). Some β-blockers that block β1AR and β2ARs can activate β1LARs at higher concentrations than those required to cause blockade. The β2AR does not form a corresponding low affinity binding site (Baker et al 2002) and therefore we postulated that heterologous amino acids are responsible for the formation of β1LAR. Our aim was to investigate whether heterologous amino acids of transmembrane domain V (TMDV) of β1AR and β2ARs contribute to β1LAR. β1ARs, β2ARs and mutant β1ARs containing all (β1(β2TMDV)AR) or single amino acids of TMDV of the β2AR were prepared and stably expressed in Chinese Hamster Ovary cells. Concentration-effect curves for cyclicAMP accumulation were carried out for (-)-CGP12177 or (-)-isoprenaline in the absence or presence of (-)-bupranolol. _______________________________________________________________________ (-)-CGP 12177 (-)-Bupranolol affinity (pKB) pEC50 vs (-)-CGP 12177 vs (-)-isoprenaline _______________________________________________________________________ β1AR 8.00 ± 0.11 (11) 7.23 ± 0.23 (5) 9.52 ± 0.28 (5) β2AR (high density) 9.24 ± 0.14 (5) 9.82 ± 0.52 (8) xPaulxxxxxxx β2AR (low density) no effect β1(β2TMV)AR 8.86 ± 0.10 (15) 8.06 ± 0.17 (8) 9.08 ± 0.22 (6) β1(V230I)AR 9.07 ± 0.07 (10) 7.64 ± 0.12 (8) 9.36 ± 0.28 (9) β1(R222Q)AR 8.09 ± 0.29 (6) 7.33 ± 0.23 (5) 9.36 ± 0.08 (6) β1(V230A)AR 7.59 ± 0.09 (6) 7.32 ± 0.24 (4) 8.62 ± 0.18 (5) _______________________________________________________________________ The potency of (-)-CGP12177 was higher at β2AR than at β1AR consistent with activation through a low affinity site at the β1AR (β1LAR) but not β2AR. The presence of V230 in β1AR accounted for the lower potency of (-)-CGP 12177. The affinity of (-)-bupranolol at β1AR and mutants was higher when determined with (-)-isoprenaline than with (-)-CGP 12177. The affinity of (-)-bupranolol determined against (-)-CGP 12177 was lower at β1AR compared to β2AR. The presence of V230 in β1AR accounted in part for the lower affinity. In conclusion V230 of the β1AR contributes in part to the low affinity binding site of β1AR. Baker JG, Hall IP, Hill SJ (2002). Pharmacological characterization of CGP12177 at the human β2-adrenoceptor. Br J Pharmacol 137, 400−408 Kaumann AJ, Molenaar P (2008) The low-affinity site of the β1-adrenoceptor and its relevance to cardiovascular pharmacology. Pharmacol Ther 118, 303-336

TaNF-YC11, one of the light-upregulated NF-YC members in Triticum aestivum, is co-regulated with photosynthesis-related genes

NF-Y is a heterotrimeric transcription factor complex. Each of the NF-Y subunits (NF-YA, NF-YB and NF-YC) in plants is encoded by multiple genes. Quantitative RT-PCR analysis revealed that five wheat NF-YC members (TaNF-YC5, 8, 9, 11 & 12) were upregulated by light in both the leaf and seedling shoot. Co-expression analysis of Affymetrix wheat genome array datasets revealed that transcript levels of a large number of genes were consistently correlated with those of the TaNF-YC11 and TaNF-YC8 genes in 3-4 separate Affymetrix array datasets. TaNF-YC11-correlated transcripts were significantly enriched with the Gene Ontology term photosynthesis. Sequence analysis in the promoters of TaNF-YC11-correlated genes revealed the presence of putative NF-Y complex binding sites (CCAAT motifs). Quantitative RT-PCR analysis of a subset of potential TaNF-YC11 target genes showed that ten out of the thirteen genes were also light-upregulated in both the leaf and seedling shoot and had significantly correlated expression profiles with TaNF-YC11. The potential target genes for TaNF-YC11 include subunit members from all four thylakoid membrane bound complexes required for the conversion of solar energy into chemical energy and rate limiting enzymes in the Calvin cycle. These data indicate that TaNF-YC11 is potentially involved in regulation of photosynthesis-related genes.

Influence of physical and chemical properties of solids on heavy metal adsorption

Partition of heavy metals between particulate and dissolve fraction of stormwater primarily depends on the adsorption characteristics of solids particles. Moreover, the bioavailability of heavy metals is also influenced by the adsorption behaviour of solids. However, due to the lack of fundamental knowledge in relation to the heavy metals adsorption processes of road deposited solids, the effectiveness of stormwater management strategies can be limited. The research study focused on the investigation of the physical and chemical parameters of solids on urban road surfaces and, more specifically, on heavy metal adsorption to solids. Due to the complex nature of heavy metal interaction with solids, a substantial database was generated through a series of field investigations and laboratory experiments. The study sites for the build-up pollutant sample collection were selected from four urbanised suburbs located in a major river catchment. Sixteen road sites were selected from these suburbs and represented typical industrial, commercial and residential land uses. Build-up pollutants were collected using a wet and dry vacuum collection technique which was specially designed to improve fine particle collection. Roadside soil samples were also collected from each suburb for comparison with the road surface solids. The collected build-up solids samples were separated into four particle size ranges and tested for a range of physical and chemical parameters. The solids build-up on road surfaces contained a high fraction (70%) of particles smaller than 150ìm, which are favourable for heavy metal adsorption. These solids particles predominantly consist of soil derived minerals which included quartz, albite, microcline, muscovite and chlorite. Additionally, a high percentage of amorphous content was also identified in road deposited solids. In comparing the mineralogical data of surrounding soil and road deposited solids, it was found that about 30% of the solids consisted of particles generated from traffic related activities on road surfaces. Significant difference in mineralogical composition was noted in different particle sizes of build-up solids. Fine solids particles (<150ìm) consisted of a clayey matrix and high amorphous content (in the region of 40%) while coarse particles (>150ìm) consisted of a sandy matrix at all study sites, with about 60% quartz content. Due to these differences in mineralogical components, particles larger than and smaller than 150ìm had significant differences in their specific surface area (SSA) and effective cation exchange capacity (ECEC). These parameters, in turn, exert a significant influence on heavy metal adsorption. Consequently, heavy metal content in >150ìm particles was lower than in the case of fine particles. The particle size range <75ìm had the highest heavy metal content, corresponding with its high clay forming minerals, high organic matter and low quartz content which increased the SSA, ECEC and the presence of Fe, Al and Mn oxides. The clay forming minerals, high organic matter and Fe, Al and Mn oxides create distinct groups of charge sites on solids surfaces and exhibit different adsorption mechanisms and bond strength, between heavy metal elements and charge sites. Therefore, the predominance of these factors in different particle sizes leads to different heavy metal adsorption characteristics. Heavy metals show preference for association with clay forming minerals in fine solids particles, whilst in coarse particles heavy metals preferentially associate with organic matter. Although heavy metal adsorption to amorphous material is very low, the heavy metals embedded in traffic related materials have a potential impact on stormwater quality.Adsorption of heavy metals is not confined to an individual type of charge site in solids, whereas specific heavy metal elements show preference for adsorption to several different types of charge sites in solids. This is attributed to the dearth of preferred binding sites and the inability to reach the preferred binding sites due to competition between different heavy metal species. This confirms that heavy metal adsorption is significantly influenced by the physical and chemical parameters of solids that lead to a heterogeneity of surface charge sites. The research study highlighted the importance of removal of solids particles from stormwater runoff before they enter into receiving waters to reduce the potential risk posed by the bioavailability of heavy metals. The bioavailability of heavy metals not only results from the easily mobile fraction bound to the solids particles, but can also occur as a result of the dissolution of other forms of bonds by chemical changes in stormwater or microbial activity. Due to the diversity in the composition of the different particle sizes of solids and the characteristics and amount of charge sites on the particle surfaces, investigations using bulk solids are not adequate to gain an understanding of the heavy metal adsorption processes of solids particles. Therefore, the investigation of different particle size ranges is recommended for enhancing stormwater quality management practices.

Characterisation of the TaNF-Y family of transcription factors in wheat

Light plays a unique role for plants as it is both a source of energy for growth and a signal for development. Light captured by the pigments in the light harvesting complexes is used to drive the synthesis of the chemical energy required for carbon assimilation. The light perceived by photoreceptors activates effectors, such as transcription factors (TFs), which modulate the expression of light-responsive genes. Recently, it has been speculated that increasing the photosynthetic rate could further improve the yield potential of three carbon (C3) crops such as wheat. However, little is currently known about the transcriptional regulation of photosynthesis genes, particularly in crop species. Nuclear factor Y (NF-Y) TF is a functionally diverse regulator of growth and development in the model plant species, with demonstrated roles in embryo development, stress response, flowering time and chloroplast biogenesis. Furthermore, a light-responsive NF-Y binding site (CCAAT-box) is present in the promoter of a spinach photosynthesis gene. As photosynthesis genes are co-regulated by light and co-regulated genes typically have similar regulatory elements in their promoters, it seems likely that other photosynthesis genes would also have light-responsive CCAAT-boxes. This provided the impetus to investigate the NF-Y TF in bread wheat. This thesis is focussed on wheat NF-Y members that have roles in light-mediated gene regulation with an emphasis on their involvement in the regulation of photosynthesis genes. NF-Y is a heterotrimeric complex, comprised of the three subunits NF-YA, NF-YB and NF-YC. Unlike the mammalian and yeast counterparts, each of the three subunits is encoded by multiple genes in Arabidopsis. The initial step taken in this study was the identification of the wheat NF-Y family (Chapter 3). A search of the current wheat nucleotide sequence databases identified 37 NF-Y genes (10 NF-YA, 11 NF-YB, 14 NF-YC & 2 Dr1). Phylogenetic analysis revealed that each of the three wheat NF-Y (TaNF-Y) subunit families could be divided into 4-5 clades based on their conserved core regions. Outside of the core regions, eleven motifs were identified to be conserved between Arabidopsis, rice and wheat NF-Y subunit members. The expression profiles of TaNF-Y genes were constructed using quantitative real-time polymerase chain reaction (RT-PCR). Some TaNF-Y subunit members had little variation in their transcript levels among the organs, while others displayed organ-predominant expression profiles, including those expressed mainly in the photosynthetic organs. To investigate their potential role in light-mediated gene regulation, the light responsiveness of the TaNF-Y genes were examined (Chapters 4 and 5). Two TaNF-YB and five TaNF-YC members were markedly upregulated by light in both the wheat leaves and seedling shoots. To identify the potential target genes of the light-upregulated NF-Y subunit members, a gene expression correlation analysis was conducted using publically available Affymetrix Wheat Genome Array datasets. This analysis revealed that the transcript expression levels of TaNF-YB3 and TaNF-YC11 were significantly correlated with those of photosynthesis genes. These correlated express profiles were also observed in the quantitative RT-PCR dataset from wheat plants grown under light and dark conditions. Sequence analysis of the promoters of these wheat photosynthesis genes revealed that they were enriched with potential NF-Y binding sites (CCAAT-box). The potential role of TaNF-YB3 in the regulation of photosynthetic genes was further investigated using a transgenic approach (Chapter 5). Transgenic wheat lines constitutively expressing TaNF-YB3 were found to have significantly increased expression levels of photosynthesis genes, including those encoding light harvesting chlorophyll a/b-binding proteins, photosystem I reaction centre subunits, a chloroplast ATP synthase subunit and glutamyl-tRNA reductase (GluTR). GluTR is a rate-limiting enzyme in the chlorophyll biosynthesis pathway. In association with the increased expression of the photosynthesis genes, the transgenic lines had a higher leaf chlorophyll content, increased photosynthetic rate and had a more rapid early growth rate compared to the wild-type wheat. In addition to its role in the regulation of photosynthesis genes, TaNF-YB3 overexpression lines flower on average 2-days earlier than the wild-type (Chapter 6). Quantitative RT-PCR analysis showed that there was a 13-fold increase in the expression level of the floral integrator, TaFT. The transcript levels of other downstream genes (TaFT2 and TaVRN1) were also increased in the transgenic lines. Furthermore, the transcript levels of TaNF-YB3 were significantly correlated with those of constans (CO), constans-like (COL) and timing of chlorophyll a/b-binding (CAB) expression 1 [TOC1; (CCT)] domain-containing proteins known to be involved in the regulation of flowering time. To summarise the key findings of this study, 37 NF-Y genes were identified in the crop species wheat. An in depth analysis of TaNF-Y gene expression profiles revealed that the potential role of some light-upregulated members was in the regulation of photosynthetic genes. The involvement of TaNF-YB3 in the regulation of photosynthesis genes was supported by data obtained from transgenic wheat lines with increased constitutive expression of TaNF-YB3. The overexpression of TaNF-YB3 in the transgenic lines revealed this NF-YB member is also involved in the fine-tuning of flowering time. These data suggest that the NF-Y TF plays an important role in light-mediated gene regulation in wheat.

Inhibition of guinea-pig renal [Na + K]-ATPase by normotensive human plasma : effects of a high sodium diet

The effect of plasma taken from normotensive humans, while on a low and high sodium diet, on [Na + K]-ATPase and 3H-ouabain binding was measured in tubules from guinea-pig kidneys. Plasma from the high sodium, compared to the low sodium, diet period: (a) inhibited [Na + K]-ATPase activity; (b) decreased 3H-ouabain affinity for binding sites; (c) increased the number of available 3H-ouabain binding sites; (d) decreased [Na + K]-ATPase turnover (activity/3H-ouabain binding sites). The inhibition of [Na + K]-ATPase suggests an increase in a (possible) natriuretic factor. The decreased affinity of 3H-ouabain binding suggests an endogenous ouabainoid, which may be the natriuretic factor.

A two-metal ion mechanism operates in the hammerhead ribozyme-mediated cleavage of an RNA substrate

Evidence for a two-metal ion mechanism for cleavage of the HH16 hammerhead ribozyme is provided by monitoring the rate of cleavage of the RNA substrate as a function of La3+ concentration in the presence of a constant concentration of Mg2+. We show that a bell-shaped curve of cleavage activation is obtained as La3+ is added in micromolar concentrations in the presence of 8 mM Mg2+, with a maximal rate of cleavage being attained in the presence of 3 microM La3+. These results show that two-metal ion binding sites on the ribozyme regulate the rate of the cleavage reaction and, on the basis of earlier estimates of the Kd values for Mg2+ of 3.5 mM and > 50 mM, that these sites bind La3+ with estimated Kd values of 0.9 and > 37.5 microM, respectively. Furthermore, given the very different effects of these metal ions at the two binding sites, with displacement of Mg2+ by La3+ at the stronger (relative to Mg2+) binding site activating catalysis and displacement of Mg2+ by La3+ at the weaker (relative to Mg2+) (relative to Mg2+) binding site inhibiting catalysis, we show that the metal ions at these two sites play very different roles. We argue that the metal ion at binding site 1 coordinates the attacking 2'-oxygen species in the reaction and lowers the pKa of the attached proton, thereby increasing the concentration of the attacking alkoxide nucleophile in an equilibrium process. In contrast, the role of the metal ion at binding site 2 is to catalyze the reaction by absorbing the negative charge that accumulates at the leaving 5'-oxygen in the transition state. We suggest structural reasons why the Mg(2+)-La3+ ion combination is particularly suited to demonstrating these different roles of the two-metal ions in the ribozyme cleavage reaction.