The matricellular protein SPARC is internalized in Sertoli, Leydig, and germ cells during testis differentiation

The gene encoding the matricellular protein secreted protein, acidic and rich in cysteine (SPARC) was identified in a screen for genes expressed sex-specifically during mouse gonad development, as being strongly upregulated in the male gonad from very early in testis development. We present here a detailed analysis of SPARC gene and protein expression during testis development, from 11.5 to 15.5 days post coitum (dpc). Section in situ hybridization analysis revealed that SPARC mRNA is expressed by the Sertoli cells in the testis cords and the fetal Leydig cells, found within the interstitial space between the testis cords. Immunodetection with anti-SPARC antibody showed that the protein was located inside the testis cords, within the cytoplasm of Sertoli and germ cells. In the interstitium, SPARC was present intracellularly within the Leydig cells. The internalization of SPARC in Sertoli, Leydig, and germ cells suggests that it plays an intracellular regulatory role in these cell types during fetal testis development.

The mitogenic signaling pathway for fibroblast growth factor-2 involves the tyrosine phosphorylation of cyclin D2 in MCF-7 human breast cancer cells

Fibroblast growth factor-2 (FGF-2) is mitogenic for the human breast cancer cell line MCF-7; here we investigate some of the signaling pathways subserving this activity. FGF-2 stimulation of MCF-7 cells resulted in a global increase of intracellular tyrosine phosphorylation of proteins, particularly FGF receptor substrate-2, the protooncogene product Src and the mitogen-activated protein kinase (MAP kinase) cascade, A major increase in the tyrosine phosphorylation of a 30-kDa protein species was also found. This protein was identified as cyclin D2 by mass spectrometry after trypsin digestion. Immunoprecipitation of cyclin D2 and immunoblotting with anti-phosphotyrosine antibodies confirmed that the tyrosine phosphorylation of cyclin D2 was indeed induced by FGF-2 stimulation. In addition, pharmacological inhibition of Src (with herbimycin A and PP2), and of the MAP kinase cascade (with PD98059), confirmed that Src activity is required for the FGF-2-induced phosphorylation of cyclin D2 whereas MAP kinase activity is not, Thus, tyrosine phosphorylation of cyclin D2 may be a hey regulatory target for FGF-2 signaling. (C) 2000 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

Expression of apoptotic regulatory molecules in renal cell carcinoma: Elevated expression of Fas ligand

Renal cell carcinoma (RCC) is the most common renal neoplasm. Despite being infiltrated by tumour infiltrating lymphocytes (TIL), these TIL are unable to control tumour growth in vivo, suggesting that the cytotoxic capacity of TIL against RCC is impaired, or that the tumour cells are resistant to killing and therefore escape detection by the immune system. It is postulated that the expression of apoptotic regulatory molecules in RCC favours tumour cell survival. The present study has therefore determined the expression of Fas (APO- 1/CD95), Fas ligand (Fas L) and bcl-2 in these tumours. The expression of Fas, Fas L and bcl-2 mRNA transcripts was determined in RCC, normal kidney and peripheral blood by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR), following RNA extraction and cDNA synthesis from tissues and cell samples. Transcript levels were measured by densitometry after Southern blot hybridization of PCR products with internal radio-labelled oligonucleotide probes; a densitometry score was assigned to each hybridizing DNA band and expressed as a ratio of the glyceraldehyde-3-phosphate dehydrogenase content. In peripheral blood, the expression of Fas L and bcl-2 transcripts was similar between patients and normal healthy individuals; however, Fas transcript expression was significantly down-regulated in the patients' versus normal peripheral blood (P = 0.026). Most interestingly, significantly up-regulated Fas L expression was observed in RCC compared to normal kidney (P = 0.041). In contrast, bcl-2 transcripts were well represented in normal kidney but markedly decreased in RCC (P = 0.021). The expression of Fas transcripts in normal kidney and RCC was variable. These data demonstrate elevated expression of Fas L transcripts in RCC, but the functional relevance of this remains to be investigated.

Evolving noisy oscillatory dynamics in genetic regulatory networks

We introduce a genetic programming (GP) approach for evolving genetic networks that demonstrate desired dynamics when simulated as a discrete stochastic process. Our representation of genetic networks is based on a biochemical reaction model including key elements such as transcription, translation and post-translational modifications. The stochastic, reaction-based GP system is similar but not identical with algorithmic chemistries. We evolved genetic networks with noisy oscillatory dynamics. The results show the practicality of evolving particular dynamics in gene regulatory networks when modelled with intrinsic noise.

Effects of arachidonic acid upon the volume-sensitive chloride current in rat osteoblast-like (ROS 17/2.8) cells.

1. The effects of arachidonic acid upon the volume-sensitive Cl- current present in cultured osteoblastic cells (ROS 17/2.8) was studied using the whole-cell patch-clamp technique. 2. Arachidonate produced two distinct phases of inhibition, a rapid phase occurring within 10-15 s of application preceding a slower phase that occurred 2 min after onset of arachidonate superfusion. Accompanying the slower inhibitory phase was an acceleration of the time-dependent inactivation exhibited by the current at strongly depolarized potentials (> + 50 mV). The half-maximal inhibitory concentrations (IC50) were 177 +/- 31 and 10 +/- 4 microM for the two phases respectively. 3. Arachidonate was still effective in the presence of inhibitors of cyclo-oxygenase (indomethacin, 10 microM), lipoxygenase (nordihydroguaretic acid, 10-100 microM) and cytochrome P450 (SKF525A, 100 microM; ethoxyresorufin, 10 microM; metyrapone, 500 microM; piperonyl butoxide, 500 microM; cimetidine, 1 mM). The effects of arachidonate could not be produced by another cis unsaturated fatty acid, oleic acid. 4. Measurements of cell volume showed that arachidonate effectively inhibited the regulatory volume decrease elicited by ROS 17/2.8 cells in response to a reduction in extracellular osmolarity. 5. It is concluded that the volume-sensitive Cl- conductance in ROS 17/2.8 cells is directly modulated by arachidonate and may represent a physiological mechanism by which volume regulation can be controlled in these cells.

Thermodynamics of binding of regulatory ligands to tissue transglutaminase

The transamidating activity of tissue transglutaminase is regulated by the ligands calcium and GTP, via conformational changes which facilitate or interfere with interaction with the peptidyl-glutamine substrate. We have analysed binding of these ligands by calorimetric and computational approaches. In the case of GTP we have detected a single high affinity site (K (D) approximately 1 muM), with moderate thermal effects suggestive that binding GTP involves replacement of GDP, normally bound to the protein. On line with this possibility no significant binding was observed during titration with GDP and computational studies support this view. Titration with calcium at a high cation molar excess yielded a complex binding isotherm with a number of "apparent binding sites" in large excess over those detectable by equilibrium dialysis (6 sites). This binding pattern is ascribed to occurrence of additional thermal contributions, beyond those of binding, due to the occurrence of conformational changes and to catalysis itself (with protein self-crosslinking). In contrast only one site for binding calcium with high affinity (K (D) approximately 0.15 muM) is observed with samples of enzyme inactivated by alkylation at the active site (to prevent enzyme crosslinkage and thermal effects of catalysis). These results indicate an intrinsic ability of tissue transglutaminase to bind calcium with high affinity and the necessity of careful reassessment of the enzyme regulatory pattern in relation to the concentrations of ligands in living cells, taking also in account effects of ligands on protein subcellular compartimentation.

Intracellar signalling in the HGT-1 gastric cell line and in rat isolated parietal cells

The work presented in this thesis was undertaken to increase understanding of the intracellular mechanisms regulating acid secretion by gastric parietal cells. Investigation of the effects of protein kinase C on secretory activity induced by a variety of agents was a major objective. A further aim was to establish the sites at which epidermal growth factor (EGF) acts to stimulate prostaglandin E2 (PGE2) production and to inhibit acid secretion. These investigations were carried out by using the HGT-1 human gastric cancer cell line and freshly isolated rat parietal cells. In HGT-1 cells, the cyclic AMP response to histamine and to truncated glucagon-like peptide 1 (TGLP-1) was reduced when protein kinase C was activated by 12-0-tetradecanoylphorbol 13-acetate (TPA). Receptor-binding studies and experiments in which cyclic AMP production in HGT-1 cells was stimulated by gastric inhibitory polypeptide, cholera toxin and forskolin suggested that the effect of TPA was mediated by uncoupling of the histamine H2 receptor from the guanine nucleotide regulatory protein Gs, possibly by phosphorylation of the receptor. An involvement of protein kinase C α in this effect was suggested because an antibody to this isoform specifically prevented the inhibitory effects of TPA on histamine-stimulated adenylate cyclase activity in a membrane fraction prepared from HGT-1 cells. Carbachol-stimulated secretory activity in parietal cells was specifically inhibited by Ro 31-8220, a bisindolylmaleimide inhibitor of protein kinase C. Thus protein kinase C may play a role in the activation of the secretory response to carbachol. In parietal cells prelabelled with [3H]-arachidonic acid or [3H]myristic acid, EGF did not affect [3H]-fatty acid or [3H] - diacylglycerol content. No evidence for effects of EGF on phosphatidylinositol glycan-specific phospholipase C, phospholipase A2 or on low Km cyclic AMP phosphodiesterase activities were found.

Role of the transgenic human thyrotropin receptor A-subunit in thyroiditis induced by A-subunit immunization and regulatory T cell depletion

Transgenic BALB/c mice that express intrathyroidal human thyroid stimulating hormone receptor (TSHR) A-subunit, unlike wild-type (WT) littermates, develop thyroid lymphocytic infiltration and spreading to other thyroid autoantigens after T regulatory cell (Treg) depletion and immunization with human thyrotropin receptor (hTSHR) adenovirus. To determine if this process involves intramolecular epitope spreading, we studied antibody and T cell recognition of TSHR ectodomain peptides (A–Z). In transgenic and WT mice, regardless of Treg depletion, TSHR antibodies bound predominantly to N-terminal peptide A and much less to a few downstream peptides. After Treg depletion, splenocytes from WT mice responded to peptides C, D and J (all in the A-subunit), but transgenic splenocytes recognized only peptide D. Because CD4+ T cells are critical for thyroid lymphocytic infiltration, amino acid sequences of these peptides were examined for in silico binding to BALB/c major histocompatibility complex class II (IA–d). High affinity subsequences (inhibitory concentration of 50% < 50 nm) are present in peptides C and D (not J) of the hTSHR and mouse TSHR equivalents. These data probably explain why transgenic splenocytes do not recognize peptide J. Mouse TSHR mRNA levels are comparable in transgenic and WT thyroids, but only transgenics have human A-subunit mRNA. Transgenic mice can present mouse TSHR and human A-subunit-derived peptides. However, WT mice can present only mouse TSHR, and two to four amino acid species differences may preclude recognition by CD4+ T cells activated by hTSHR-adenovirus. Overall, thyroid lymphocytic infiltration in the transgenic mice is unrelated to epitopic spreading but involves human A-subunit peptides for recognition by T cells activated using the hTSHR.

Sustained insulin secretory response in human islets co-cultured with pancreatic duct-derived epithelial cells within a rotational cell culture system

Aims/hypothesis - Loss of the trophic support provided by surrounding non-endocrine pancreatic cell populations underlies the decline in beta cell mass and insulin secretory function observed in human islets following isolation and culture. This study sought to determine whether restoration of regulatory influences mediated by ductal epithelial cells promotes sustained beta cell function in vitro. Methods - Human islets were isolated according to existing protocols. Ductal epithelial cells were harvested from the exocrine tissue remaining after islet isolation, expanded in monolayer culture and characterised using fluorescence immunocytochemistry. The two cell types were co-cultured under conventional static culture conditions or within a rotational cell culture system. The effect of co-culture on islet structural integrity, beta cell mass and insulin secretory capacity was observed for 10 days following isolation. Results - Human islets maintained under conventional culture conditions exhibited a characteristic loss in structural integrity and functional viability as indicated by a diminution of glucose responsiveness. By contrast, co-culture of islets with ductal epithelial cells led to preserved islet morphology and sustained beta cell function, most evident in co-cultures held within the rotational cell culture system, which showed a significantly (p<0.05) greater insulin secretory response to elevated glucose compared with control islets. Similarly, insulin/protein ratio data suggested that the presence of ductal epithelial cells is beneficial for the maintenance of beta cell mass. Conclusions/interpretation - The data indicate a supportive role for ductal epithelial cells in islet viability. Further characterisation of the regulatory influences may lead to novel strategies to improve long-term beta cell function both in vitro and following islet transplantation.

Expansion, harvest and cryopreservation of human mesenchymal stem cells in a serum-free microcarrier process

Human mesenchymal stem cell (hMSC) therapies are currently progressing through clinical development, driving the need for consistent, and cost effective manufacturing processes to meet the lot-sizes required for commercial production. The use of animal-derived serum is common in hMSC culture but has many drawbacks such as limited supply, lot-to-lot variability, increased regulatory burden, possibility of pathogen transmission, and reduced scope for process optimization. These constraints may impact the development of a consistent large-scale process and therefore must be addressed. The aim of this work was therefore to run a pilot study in the systematic development of serum-free hMSC manufacturing process. Human bone-marrow derived hMSCs were expanded on fibronectin-coated, non-porous plastic microcarriers in 100mL stirred spinner flasks at a density of 3×10⁵cells.mL^-1 in serum-free medium. The hMSCs were successfully harvested by our recently-developed technique using animal-free enzymatic cell detachment accompanied by agitation followed by filtration to separate the hMSCs from microcarriers, with a post-harvest viability of 99.63±0.03%. The hMSCs were found to be in accordance with the ISCT characterization criteria and maintained hMSC outgrowth and colony-forming potential. The hMSCs were held in suspension post-harvest to simulate a typical pooling time for a scaled expansion process and cryopreserved in a serum-free vehicle solution using a controlled-rate freezing process. Post-thaw viability was 75.8±1.4% with a similar 3h attachment efficiency also observed, indicating successful hMSC recovery, and attachment. This approach therefore demonstrates that once an hMSC line and appropriate medium have been selected for production, multiple unit operations can be integrated to generate an animal component-free hMSC production process from expansion through to cryopreservation.

A study of the regulatory role of retinoic acid receptor gamma in Zebrafish development

Retinoic acid (RA) is thought to signal through retinoic acid receptors (RARs), i.e. RARα, β, and γ to play important roles in embryonic development and tissue regeneration. In this thesis, the zebrafish (Danio rario) was used as a vertebrate model organism to examine the role of RARγ. Treatment of zebrafish embryos with a RARγ specific agonist reduced the axial length of developing embryos, associated with reduced somite number and loss of hoxb13a expression. There were no clear alterations in hoxc11a or myoD expression. Treatment with the RARγ agonist disrupted the formation of anterior structures of the head, the cranial bones and the anterior lateral line ganglia, associated with a loss of sox9 immunopositive cells in the same regions. Pectoral fin outgrowth was blocked by treatment with the RARγ agonist; however, this was not associated with loss of tbx5a immunopositive lateral plate cells and was reversed by wash out of the RARγ agonist or co-treatment with a RARγ antagonist. Regeneration of the transected caudal fin was also blocked by RARγ agonist treatment and restored by agonist washout or antagonist co-treatment; this phenotype was associated with a localised reduction in canonical Wnt signalling. Conversely, elevated canonical Wnt signalling after RARγ treatment was seen in other tissues, including ectopically in the notochord. Furthermore, some phenotypes seen in the RARγ treated embryos were present in mutant zebrafish embryos in which canonical Wnt signalling was constitutively increased. These data suggest that RARγ plays an essential role in maintaining neural crest and mesodermal stem/progenitor cells during normal embryonic development and tissue regeneration when the receptor is in its non-ligated state. In addition, this work has provided evidence that the activation status of RARγ may regulate hoxb13a gene expression and canonical Wnt signalling. Further research is required to confirm such novel regulatory roles.

A novel regulatory role for tissue transglutaminase in epithelial-mesenchymal transition in cystic fibrosis

Cystic fibrosis (CF) is a genetic disorder caused by mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) for which there is no overall effective treatment. Recent work indicates tissue transglutaminase (TG2) plays a pivotal intracellular role in proteostasis in CF epithelia and that the pan TG inhibitor cysteamine improves CFTR stability. Here we show TG2 has another role in CF pathology linked with TGFβ1 activation and signalling, induction of epithelial-mesenchymal transition (EMT), CFTR stability and induction of matrix deposition. We show that increased TG2 expression in normal and CF bronchial epithelial cells increases TGFβ1 levels, promoting EMT progression, and impairs tight junctions as measured by Transepithelial Electric Resistance (TEER) which can be reversed by selective inhibition of TG2 with an observed increase in CFTR stability. Our data indicate that selective inhibition of TG2 provides a potential therapeutic avenue for reducing fibrosis and increasing CFTR stability in CF.

Human Synaptic Plasticity Gene Expression Profile and Dendritic Spine Density Changes in HIV-Infected Human CNS Cells: Role in HIV-Associated Neurocognitive Disorders (HAND)

HIV-associated neurocognitive disorders (HAND) is characterized by development of cognitive, behavioral and motor abnormalities, and occur in approximately 50% of HIV infected individuals. Our current understanding of HAND emanates mainly from HIV-1 subtype B (clade B), which is prevalent in USA and Western countries. However very little information is available on neuropathogenesis of HIV-1 subtype C (clade C) that exists in Sub-Saharan Africa and Asia. Therefore, studies to identify specific neuropathogenic mechanisms associated with HAND are worth pursuing to dissect the mechanisms underlying this modulation and to prevent HAND particularly in clade B infection. In this study, we have investigated 84 key human synaptic plasticity genes differential expression profile in clade B and clade C infected primary human astrocytes by using RT2 Profile PCR Array human Synaptic Plasticity kit. Among these, 31 and 21 synaptic genes were significantly (≥3 fold) down-regulated and 5 genes were significantly (≥3 fold) up-regulated in clade B and clade C infected cells, respectively compared to the uninfected control astrocytes. In flow-cytometry analysis, down-regulation of postsynaptic density and dendrite spine morphology regulatory proteins (ARC, NMDAR1 and GRM1) was confirmed in both clade B and C infected primary human astrocytes and SK-N-MC neuroblastoma cells. Further, spine density and dendrite morphology changes by confocal microscopic analysis indicates significantly decreased spine density, loss of spines and decreased dendrite diameter, total dendrite and spine area in clade B infected SK-N-MC neuroblastoma cells compared to uninfected and clade C infected cells. We have also observed that, in clade B infected astrocytes, induction of apoptosis was significantly higher than in the clade C infected astrocytes. In conclusion, this study suggests that down-regulation of synaptic plasticity genes, decreased dendritic spine density and induction of apoptosis in astrocytes may contribute to the severe neuropathogenesis in clade B infection.

The Role of PP2A Regulatory Subunit B56 in Dictyostelium Discoideum Motility

Protein Phosphatase 2A, PP2A, is a heterotrimeric threonine/serine phosphatase system that is involved in a variety of cellular processes. This phosphatase is composed ofthree subunits: a catalytic subunit (C subunit), a scaffolding subunit (A subunit), and a regulatory subunit (B subunit). The regulatory subunit B is divided into four subclasses, B, B' (B56), B'' and B'' '. Studies showed that PP2A/B56 complexes regulate development of Dictyostelium and other metazoan cells. In addition to development, our experimental data suggest that PP2A/B56 complex also plays an important role in Dictyostelium cell motility. Cells lacking B56 was generated previously in our laboratory (Lee et al., 2008). Further studies showed that b56- cells are compromised in random cell motility compared to the wild type (AX3) cells. In contrast, b56 cells with re-introduced B56 displayed wild-type like motilities. Furthermore, one of the colleagues in our laboratory found that one of the Dictyostelium Ras species, RasG, associates with PP2A/B56 complex and RasG activation is compromised in b56- cells. Considering that Ras proteins are central in cellular motility regulation, PP2A/B56 complex may modulate cell motility through regulating Ras. We propose to determine if an introduction of constitutive active RasG proteins improves compromised b56- cell motility.

NFAT5 genes are part of the osmotic regulatory system in Atlantic salmon (Salmo salar)

Acknowledgements This study was supported by a grant from the Biotechnology and Biological Sciences Research Council (BBSRC, BB/H008063/1), UK to DGH and SAM. Funding also came from Research Council Norway for project number 241016 for DGH and EJ. This work was carried out as part of a PhD thesis funded by the Marine Alliance of Science and Technology Scotland (MASTS).