The role of oxidative stress as an underlying mechanism in life-history trade-offs

A key aspect underpinning life-history theory is the existence of trade-offs. Trade-offs occur because resources are limited, meaning that individuals cannot invest in all traits simultaneously, leading to costs for traits such as growth and reproduction. Such costs may be the reason for the sub-maximal growth rates that are often observed in nature, though the fitness consequences of these costs would depend on the effects on lifetime reproductive success. Recently, much attention has been given to the physiological mechanism that might underlie these life-history trade-offs, with oxidative stress (OS) playing a key role. OS is characterised by a build-up of oxidative damage to tissues (e.g. protein, lipids and DNA) from attack by reactive species (RS). RS, the majority of which are by-products of metabolism, are usually neutralised by antioxidants, however OS occurs when there is an imbalance between the two. There are two main theories linking OS with growth and reproduction. The first is that traits like growth and reproduction, being metabolically demanding, lead to an increase in RS production. The second involves the diversion of resources away from self-maintenance processes (e.g. the redox system) when individuals are faced with enhanced growth or reproductive expenditure. Previous research investigating trade-offs involving growth or reproduction and self-maintenance has been equivocal. One reason for this could be that associations among redox biomarkers can vary greatly so that the biomarker selected for analysis can influence the conclusion reached about an individual’s oxidative status. Therefore the first aim of my thesis was to explore the strength and pattern of integration of five biomarkers of OS (three antioxidants, one damage and one general oxidation measure) in wild blue tit (Cyanistes caeruleus) adults and nestlings (Chapter 2). In doing so, I established that all five biomarkers should be included in future analyses, thus using this collection of biomarkers I explored my next aims; whether enhanced growth (Chapters 3 and 4) or reproductive effort (Chapter 5) can lead to increased OS levels, if these traits are traded off against self-maintenance. I accomplished these aims using both a meta-analytic and experimental approach, the latter involving manipulation of brood size in wild blue tits in order to experimentally alter growth rate of nestlings and provisioning rate (a proxy for reproductive expenditure) of adults. I also investigated the potential for redox integration to be used as an index of body condition (Chapter 2), allowing predictions about future fitness consequences of changes to oxidative state to be made. A growth – self-maintenance trade off was supported by my meta-analytic results (Chapter 4) which found OS to be a constraint on growth. However, when faced with experimentally enhanced growth, animals were typically not able to adjust this trade-off so that oxidative damage resulted. This might support the idea that energetically expensive growth causes resources to be diverted away from the redox system; however, antioxidants did not show an overall reduction in response to growth in the meta-analysis suggesting that oxidative costs of growth may result from increased RS production due to the greater metabolism needed for enhanced growth. My experimental data (Chapter 3) showed a similar pattern, with raised protein damage levels (protein carbonyls; PCs) in the fastest growing blue tit chicks in a brood, compared with their slower growing sibs. These within-brood differences in OS levels likely resulted from within-brood hierarchies and might have masked any between-brood differences, which were not observed here. Despite evidence for a growth – self-maintenance trade off, my experimental results on blue tits found no support for the hypothesis that self-maintenance is also traded off against reproduction, another energetically demanding trait. There was no link between experimentally altered reproductive expenditure and OS, nor was there a direct correlation between reproductive effort and OS (Chapter 5). However, there are various factors that likely influence whether oxidative costs are observed, including environmental conditions and whether such costs are transient. This emphasises the need for longitudinal studies following the same individuals over multiple years and across a wide range of habitats that differ in quality. This would allow investigation into how key life events interact; it might be that raised OS levels from rapid early growth have the potential to constrain reproduction or that high parental OS levels constrain offspring growth. Any oxidative costs resulting from these life-history trade-offs have the potential to impact on future fitness. Redox integration of certain biomarkers might prove to be a useful tool in making predictions about fitness, as I found in Chapter 2, as well as establishing how the redox system responds, as a whole, to changes to growth and reproduction. Finally, if the tissues measured can tolerate a given level of OS, then the level of oxidative damage might be irrelevant and not impact on future fitness at all.

The p38 mitogen-activated protein kinase regulates interleukin-1beta-induced IL-8 expression via an effect on the IL-8 promoter in intestinal epithelial cells

Several lines of evidence implicate the p38 mitogen-activated protein kinase (p38 MAPK) in the proinflammatory response to bacterial agents and cytokines. Equally, the transcription factor, nuclear factor (NF)-kappaB, is recognized to be a critical determinant of the inflammatory response in intestinal epithelial cells (IECs). However, the precise inter-relationship between the activation of p38 MAPK and activation of the transcription factor NF-kappaB in the intestinal epithelial cell (IEC) system, remains unknown. Here we show that interleukin (IL)-1beta activates all three MAPKs in Caco-2 cells. The production of IL-8 and monocyte chemotactic protein 1 (MCP-1) was attenuated by 50% when these cells were preincubated with the p38 MAPK inhibitor, SB 203580. Further investigation of the NF-kappaB signalling system revealed that the inhibitory effect was independent of the phosphorylation and degradation of IkappaBalpha, the binding partner of NF-kappaB. This effect was also independent of the DNA binding of the p65 Rel A subunit, as well as transactivation, determined by an NF-kappaB luciferase construct, using both SB 203580 and dominant-negative p38 MAPK. Evaluation of IL-8 and MCP-1 RNA messages by reverse transcription-polymerase chain reaction (RT-PCR) revealed that the inhibitory effect of SB 203580 was associated with a reduction in this parameter. Using an IL-8-luciferase promoter construct, an effect of p38 upon its activation by both pharmacological and dominant-negative p38 construct co-transfection was demonstrated. It is concluded that p38 MAPK influences the expression of chemokines in intestinal epithelial cells, through an effect upon the activation of the chemokine promoter, and does not directly involve the activation of the transcription factor NF-kappaB

INTS3 controls the hSSB1-mediated DNA damage response

Human SSB1 (single-stranded binding protein 1 [hSSB1]) was recently identified as a part of the ataxia telangiectasia mutated (ATM) signaling pathway. To investigate hSSB1 function, we performed tandem affinity purifications of hSSB1 mutants mimicking the unphosphorylated and ATM-phosphorylated states. Both hSSB1 mutants copurified a subset of Integrator complex subunits and the uncharacterized protein LOC58493/c9orf80 (henceforth minute INTS3/hSSB-associated element [MISE]). The INTS3–MISE–hSSB1 complex plays a key role in ATM activation and RAD51 recruitment to DNA damage foci during the response to genotoxic stresses. These effects on the DNA damage response are caused by the control of hSSB1 transcription via INTS3, demonstrating a new network controlling hSSB1 function.

Porphyromonas gingivalis lipopolysaccharide alters atherosclerotic-related gene expression in oxidized low-density-lipoprotein-induced macrophages and foam cells

The molecular mechanism between atherosclerosis formation and periodontal pathogens is not clear although positive correlation between periodontal infections and cardiovascular diseases has been reported. Objective: To determine if atherosclerosis related genes were affected in foam cells during and after its formation by P. gingivalis lipopolysaccharide (LPS) stimulation. Methods: Macrophages from human THP-1 monocytes were treated with oxidized low density lipoprotein (oxLDL) to induce the formation of foam cells. P. gingivalis LPS was added to cultures of either oxLDL-induced macrophages or foam cells. The expression of atherosclerosis related genes was assayed by quantitative real time PCR and the protein production of granulocyte-macrophage colony-stimulating factor（GM-CSF）, monocyte chemotactic protein-1 (MCP-1), IL-1β, IL-10 and IL-12 was determined by ELISA. Nuclear translocation of NF-κB P65 was detected by immunocytochemistry and western blot was used to evaluate IKB-α degradation to confirm the NF-κB pathway activation. Results: P. gingivalis LPS stimulated atherosclerosis related gene expression in foam cells and increased oxLDL induced expression of chemokines, adhesion molecules, growth factors, apoptotic genes, and nuclear receptors in macrophages. Transcription of the pro-inflammatory cytokines IL-1β and IL-12 was elevated in response to LPS in both macrophages and foam cells, whereas the anti-inflammatory cytokine IL-10 was not affected. Increased NF-κB pathway activation was also observed in LPS and oxLDL co-stimulated macrophages. Conclusion: P. gingivalis LPS appears to be an important factor in the development of atherosclerosis by stimulation of atherosclerosis related gene expression in both macrophages and foam cells via activation of the NF-κB pathway.

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.

Clusterin is a critical downstream mediator of stress-induced YB-1 transactivation in prostate cancer

Clusterin is a stress-activated, cytoprotective chaperone that confers broad-spectrum treatment resistance in cancer. However, the molecular mechanisms mediating CLU transcription following anticancer treatment stress remain incompletely defined. We report that Y-box binding protein-1 (YB-1) directly binds to CLU promoter regions to transcriptionally regulate clusterin expression. In response to endoplasmic reticulum stress inducers, including paclitaxel, YB-1 is translocated to the nucleus to transactivate clusterin. Furthermore, higher levels of activated YB-1 and clusterin are seen in taxane-resistant, compared with parental, prostate cancer cells. Knockdown of either YB-1 or clusterin sensitized prostate cancer cells to paclitaxel, whereas their overexpression increased resistance to taxane. Clusterin overexpression rescued cells from increased paclitaxel-induced apoptosis following YB-1 knockdown; in contrast, however, YB-1 overexpression did not rescue cells from increased paclitaxel-induced apoptosis following clusterin knockdown. Collectively, these data indicate that YB-1 transactivation of clusterin in response to stress is a critical mediator of paclitaxel resistance in prostate cancer. Mol Cancer Res; 9(12); 1755–66.

A potential role for lamellar insulin-like growth factor-1 receptor in the pathogenesis of hyperinsulinaemic laminitis

The reason why a sustained high concentration of insulin induces laminitis in horses remains unclear. Cell proliferation occurs in the lamellae during insulin-induced laminitis and in other species high concentrations of insulin can activate receptors for the powerful cell mitogen, insulin-like growth factor (IGF)-1. The first aim of this study was to determine if IGF-1 receptors (IGF-1R) are activated in the hoof during insulin-induced laminitis. Gene expression for IGF-1R and the insulin receptor (InsR) was measured using qRT-PCR, in lamellar tissue from control horses and from horses undergoing a prolonged euglycaemic, hyperinsulinaemic clamp (p-EHC), during the mid-developmental (24 h) and acute (46 h) phases of insulin-induced laminitis. Gene expression for both receptors was decreased 13–32-fold (P < 0.05) at both time-points in the insulin-treated horses. A second aim was to determine if the down-regulation of the receptor genes could be accounted for by an increase in circulating IGF-1. Serum IGF-1 was measured at 0, 10, 25 and 46 h post-treatment in horses given a p-EHC for approximately 46 h, and in matched controls administered a balanced, electrolyte solution. There was no increase in serum IGF-1 concentrations during the p-EHC, consistent with down-regulation of both receptors by insulin. Stimulation of the IGF-1R by insulin may lead to inappropriate lamellar epidermal cell proliferation and lamellar weakening, a potential mechanism for hyperinsulinaemic laminitis. Targeting this receptor may provide insights into the pathogenesis or identify a novel therapy for hyperinsulinaemic laminitis.

Hyperlipidemia impaired innate immune response to periodontal pathogen Porphyromonas gingivalis in apolipoprotein E knockout mice

A finely-tuned innate immune response plays a pivotal role in protecting host against bacterial invasion during periodontal disease progression. Hyperlipidemia has been suggested to exacerbate periodontal health condition. However, the underlying mechanism has not been addressed. In the present study, we investigated the effect of hyperlipidemia on innate immune responses to periodontal pathogen Porphyromonas gingivalis infection. Apolipoprotein E-deficient and wild-type mice at the age of 20 weeks were used for the study. Peritoneal macrophages were isolated and subsequently used for the study of viable P. gingivalis infection. ApoE−/− mice demonstrated inhibited iNOS production and impaired clearance of P. gingivalis in vitro and in vivo; furthermore, ApoE−/− mice displayed disrupted cytokine production pattern in response to P. gingivalis, with a decreased production of tumor necrosis factor-α, interleukin-6 (IL-6), IL-1β and monocyte chemotactic protein-1. Microarray data demonstrated that Toll-like receptor (TLR) and NOD-like receptor (NLR) pathway were altered in ApoE−/− mice macrophages; further analysis of pattern recognition receptors (PRRs) demonstrated that expression of triggering receptors on myeloid cells-1 (TREM-1), an amplifier of the TLR and NLR pathway, was decreased in ApoE−/− mice macrophages, leading to decreased recruitment of NF-κB onto the promoters of the TNF-α and IL-6. Our data suggest that in ApoE−/− mice hyperlipidemia disrupts the expression of PRRs, and cripples the host’s capability to generate sufficient innate immune response to P. gingivalis, which may facilitate immune evasion, subgingival colonization and establishment of P. gingivalis in the periodontal niche.

Zymosan-induced inflammation stimulates neo-adipogenesis

Objective: To investigate the potential of inflammation to induce new adipose tissue formation in the in vivo environment. Methods and results: Using an established model of in vivo adipogenesis, a silicone chamber containing a Matrigel and fibroblast growth factor 2 (1 μg/ml) matrix was implanted into each groin of an adult male C57Bl6 mouse and vascularized with the inferior epigastric vessels. Sterile inflammation was induced in one of the two chambers by suspending Zymosan-A (ZA) (200-0.02 μg/ml) in the matrix at implantation. Adipose tissue formation was assessed at 6, 8, 12 and 24 weeks. ZA induced significant adipogenesis in an inverse dose-dependent manner (P<0.001). At 6 weeks adipose tissue formation was greatest with the lowest concentrations of ZA and least with the highest. Adipogenesis occurred both locally in the chamber containing ZA and in the ZA-free chamber in the contralateral groin of the same animal. ZA induced a systemic inflammatory response characterized by elevated serum tumour necrosis factor-α levels at early time points. Aminoguanidine (40 μg/ml) inhibited the adipogenic response to ZA-induced inflammation. Adipose tissue formed in response to ZA remained stable for 24 weeks, even when exposed to the normal tissue environment. Conclusions: These results demonstrate that inflammation can drive neo-adipogenesis in vivo. This suggests the existence of a positive feedback mechanism in obesity, whereby the state of chronic, low-grade inflammation, characteristic of the condition, may promote further adipogenesis. The mobilization and recruitment of a circulating population of adipose precursor cells is likely to be implicated in this mechanism.

Acute resistance exercise increases the expression of chemotactic factors within skeletal muscle

Intense resistance exercise causes mechanical loading of skeletal muscle, followed by muscle adaptation. Chemotactic factors likely play an important role in these processes. Purpose We investigated the time course of changes in the expression and tissue localization of several key chemotactic factors in skeletal muscle during the early phase of recovery following resistance exercise. Methods Muscle biopsy samples were obtained from vastus lateralis of eight untrained men (22+-0.5 yrs) before and 2, 4 and 24 h after three sets of leg press, squat and leg extension at 80% 1 RM. Results Monocyte chemotactic protein-1 (95×), interleukin-8 (2,300×), IL-6 (317×), urokinase-type plasminogen activator (15×), vascular endothelial growth factor (2×) and fractalkine (2.5×) mRNA was significantly elevated 2 h post-exercise. Interleukin-8 (38×) and interleukin-6 (58×) protein was also significantly elevated 2 h post-exercise, while monocyte chemotactic protein-1 protein was significantly elevated at 2 h (22×) and 4 h (21×) post-exercise. Monocyte chemotactic protein-1 and interleukin-8 were expressed by cells residing in the interstitial space between muscle fibers and, in some cases, were co-localized with CD68+ macrophages, PAX7+ satellite cells and blood vessels. However, the patterns of staining were inconclusive and not consistent. Conclusion In conclusion, resistance exercise stimulated a marked increase in the mRNA and protein expression of various chemotactic factors in skeletal muscle. Myofibers were not the dominant source of these factors. These findings suggest that chemotactic factors regulate remodeling/adaptation of skeletal muscle during the early phase of recovery following resistance exercise.

The ADAMTS1 protease gene is required for mammary tumor growth and metastasis

A disintegrin and metalloprotease with thrombospondin motifs protein 1 (ADAMTS1) is a protease commonly up-regulated in metastatic carcinoma. Its overexpression in cancer cells promotes experimental metastasis, but whether ADAMTS1 is essential for metastatic progression is unknown. To address this question, we investigated mammary cancer progression and spontaneous metastasis in the MMTV-PyMT mouse mammary tumor model in Adamts1 knockout mice. Adamts1−/−/PyMT mice displayed significantly reduced mammary tumor and lung metastatic tumor burden and increased survival, compared with their wild-type and heterozygous littermates. Histological examination revealed an increased proportion of tumors with ductal carcinoma in situ and a lower proportion of high-grade invasive tumors in Adamts1−/−/PyMT mice, compared with Adamts1+/+/PyMT mice. Increased apoptosis with unaltered proliferation and vascular density in the Adamts1−/−/PyMT tumors suggested that reduced cell survival accounts for the lower tumor burden in ADAMTS1-deficient mice. Furthermore, Adamts1−/− tumor stroma had significantly lesser amounts of proteolytically cleaved versican and increased numbers of CD45+ leukocytes. Characterization of immune cell gene expression indicated that cytotoxic cell activation was increased in Adamts1−/− tumors, compared with Adamts1+/+ tumors. This finding is supported by significantly elevated IL-12+ cell numbers in Adamts1−/− tumors. Thus, in vivo ADAMTS1 may promote mammary tumor growth and progression to metastasis in the PyMT model and is a potential therapeutic target to prevent metastatic breast cancer.

Evaluation of the spatial distribution of γH2AX following ionizing radiation

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX1,2. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)3. The phosphorylated form of H2AX, referred to as γH2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorecence microscopy3. Analysis and quantitation of γH2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of γH2AX as molecular marker of DSBs, a disparity in γ-irradiation-induced γH2AX foci formation in euchromatin and heterochromatin has been observed5-7. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of γH2AX following ionizing radiation8. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between γH2AX formation and active transcription9. Here we demonstrate our immunofluorescence method for detection and quantitation of γH2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3Dmodeling.

Suggestive linkage of the parathyroid receptor type 1 to osteoporosis

We have investigated the role of 23 candidate genes in the control of bone mineral density (BMD) by linkage studies in families of probands with osteoporosis (lumbar spine [LS] or femoral neck [FN] BMD T score < -2.5) and low BMD relative to an age- and gender-matched cohort (Z score < -2.0). One hundred and fifteen probands (35 male, 80 female) and 499 of their first- or second-degree relatives (223 males and 276 females) were recruited for the study. BMD was measured at the LS and FN using dual-energy X-ray absorptiometry and expressed as age- and gender-matched Z scores corrected for body mass index. The candidate genes studied were the androgen receptor, type I collagen A1 (COLIA1), COLIA2, COLIIA1, vitamin D receptor (VDR), colony-stimulating factor 1, calcium-sensing receptor, epidermal growth factor (EGF), estrogen receptor 1 (ESR1), fibrillin type 1, insulin-like growth factor 1, interleukin-1 alpha (IL-1α), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-11 (IL-11), osteopontin, parathyroid hormone (PTH), PTH-related peptide, PTH receptor type 1 (PTHR1), transforming growth factor-beta 1, and tumor necrosis factors alpha and beta. Sixty-four microsatellites lying close to or within these genes were investigated for linkage with BMD. Using the program MapMaker/Sibs there was suggestive evidence of linkage between BMD and PTHR1 (maximum LOD score obtained [MLS] 2.7-3.5). Moderate evidence of linkage was also observed with EGF (MLS 1.8), COLIA1 (MLS 1.7), COLIIA1/VDR (MLS 1.7), ESR1 (MLS 1.4), IL-1α (MLS 1.4), IL-4 (MLS 1.2), and IL-6 (MLS 1.2). Variance components analysis using the program ACT, correcting for proband-wise ascertainment, also showed evidence of linkage (p ≤0.05) at markers close to or within the candidate genes IL- 1α, PTHR1, IL-6, and COLIIA1/VDR. Further studies will be required to confirm these findings, to refine the location of gene responsible for the observed linkage, and to screen the candidate genes targeted at these loci for mutations.

Growth hormone resistance and somatomedins in children with end-stage liver disease awaiting transplantation

Background: The success of orthotopic liver transplantation as treatment for end-stage liver disease has prompted investigation of strategies to maintain or improve nutrition and growth in children awaiting transplantation, because malnutrition is an adverse prognostic factor. The purpose of this study was to evaluate the effect of recombinant human growth hormone therapy on body composition and indices of liver function in patients awaiting transplant. Methods: The study was designed as a placebo- controlled, double-blind, crossover trial. Patients received 0.2 U/kg growth hormone, subcutaneously, or placebo daily for 28 days during two treatment periods, separated by a 2-week washout period. Ten patients (mean age, 3.06 ± 1.15 years; range, 0.51-11.65 years, five men), with extrahepatic biliary atresia (n = 8) or two with Alagille's syndrome (n = 2), with end-stage liver disease, completed the trial while awaiting orthotopic liver transplantation. Height, weight, total body potassium, total body fat, resting energy expenditure, respiratory quotient, hematologic and multiple biochemical profile, number of albumin infusions, insulin-like growth factor-1 and 1, growth hormone binding protein (GHBP), and insulin-like growth factor binding protein-1 (IGFBP-1) and insulin-like growth factor binding protein (IGFBP-3) were measured at the beginning and end of each treatment period. Results: Growth hormone treatment was associated with a significant decline in serum bilirubin (-34.6 ± 16.5 μmol/l vs. 18.2 ± 11.59 μmol/l; p < 0.02) but there was no significant effect on any anthropometric or body composition measurements, or on any biochemical or hematologic parameters. Conclusions: These children with end-stage liver disease displayed growth hormone resistance, particularly in relation to the somatomedin axis. Exogenous growth hormone administration may be of limited value in these patients

Modeling chickpea growth and development: Nitrogen accumulation and use

Quantitative information regarding nitrogen (N) accumulation and its distribution to leaves, stems and grains under varying environmental and growth conditions are limited for chickpea (Cicer arietinum L.). The information is required for the development of crop growth models and also for assessment of the contribution of chickpea to N balances in cropping systems. Accordingly, these processes were quantified in chickpea under different environmental and growth conditions (still without water or N deficit) using four field experiments and 1325 N measurements. N concentration ([N]) in green leaves was 50 mg g-1 up to beginning of seed growth, and then it declined linearly to 30 mg g-1 at the end of seed growth phase. [N] in senesced leaves was 12 mg g-1. Stem [N] decreased from 30 mg g-1 early in the season to 8 mg g-1 in senesced stems at maturity. Pod [N] was constant (35 mg g-1), but grain [N] decreased from 60 mg g-1 early in seed growth to 43 mg g-1 at maturity. Total N accumulation ranged between 9 and 30 g m-2. N accumulation was closely linked to biomass accumulation until maturity. N accumulation efficiency (N accumulation relative to biomass accumulation) was 0.033 g g-1 where total biomass was -2 and during early growth period, but it decreased to 0.0176 g g-1 during the later growth period when total biomass was >218 g m-2. During vegetative growth (up to first-pod), 58% of N was partitioned to leaves and 42% to stems. Depending on growth conditions, 37-72% of leaf N and 12-56% of stem N was remobilized to the grains. The parameter estimates and functions obtained in this study can be used in chickpea simulation models to simulate N accumulation and distribution.