Secretion Of Phosphatase Research Articles

Expression of Concern: Secretory Phosphatases Deficient Mutant of Mycobacterium tuberculosis Imparts Protection at the Primary Site of Infection in Guinea Pigs.

Expression of Concern: Secretory Phosphatases Deficient Mutant of Mycobacterium tuberculosis Imparts Protection at the Primary Site of Infection in Guinea Pigs.

Plant Foraging Strategies Driven by Distinct Genetic Modules: Cross-Ecosystem Transcriptomics Approach.

Plants have evolved diverse strategies for foraging, e.g., mycorrhizae, modification of root system architecture, and secretion of phosphatase. Despite extensive molecular/physiological studies on individual strategies under laboratory/greenhouse conditions, there is little information about how plants orchestrate these strategies in the field. We hypothesized that individual strategies are independently driven by corresponding genetic modules in response to deficiency/unbalance in nutrients. Roots colonized by mycorrhizal fungi, leaves, and root-zone soils were collected from 251 maize plants grown across the United States Corn Belt and Japan, which provided a large gradient of soil characteristics/agricultural practice and thus gene expression for foraging. RNA was extracted from the roots, sequenced, and subjected to gene coexpression network analysis. Nineteen genetic modules were defined and functionally characterized, from which three genetic modules, mycorrhiza formation, phosphate starvation response (PSR), and root development, were selected as those directly involved in foraging. The mycorrhizal module consists of genes responsible for mycorrhiza formation and was upregulated by both phosphorus and nitrogen deficiencies. The PSR module that consists of genes encoding phosphate transporter, secreted acid phosphatase, and enzymes involved in internal-phosphate recycling was regulated independent of the mycorrhizal module and strongly upregulated by phosphorus deficiency relative to nitrogen. The root development module that consists of regulatory genes for root development and cellulose biogenesis was upregulated by phosphorus and nitrogen enrichment. The expression of this module was negatively correlated with that of the mycorrhizal module, suggesting that root development is intrinsically an opposite strategy of mycorrhizae. Our approach provides new insights into understanding plant foraging strategies in complex environments at the molecular level.

Effective Small Molecule Antibacterials from a Novel Anti-Protein Secretion Screen.

The increasing problem of bacterial resistance to antibiotics underscores the urgent need for new antibacterials. Protein export pathways are attractive potential targets. The Sec pathway is essential for bacterial viability and includes components that are absent from eukaryotes. Here, we used a new high-throughput in vivo screen based on the secretion and activity of alkaline phosphatase (PhoA), a Sec-dependent secreted enzyme that becomes active in the periplasm. The assay was optimized for a luminescence-based substrate and was used to screen a ~240K small molecule compound library. After hit confirmation and analoging, 14 HTS secretion inhibitors (HSI), belonging to eight structural classes, were identified with IC50 < 60 µM. The inhibitors were evaluated as antibacterials against 19 Gram-negative and Gram-positive bacterial species (including those from the WHO’s top pathogens list). Seven of them—HSI#6, 9; HSI#1, 5, 10; and HSI#12, 14—representing three structural families, were bacteriocidal. HSI#6 was the most potent hit against 13 species of both Gram-negative and Gram-positive bacteria with IC50 of 0.4 to 8.7 μM. HSI#1, 5, 9 and 10 inhibited the viability of Gram-positive bacteria with IC50 ~6.9–77.8 μM. HSI#9, 12, and 14 inhibited the viability of E. coli strains with IC50 < 65 μM. Moreover, HSI#1, 5 and 10 inhibited the viability of an E. coli strain missing TolC to improve permeability with IC50 4 to 14 μM, indicating their inability to penetrate the outer membrane. The antimicrobial activity was not related to the inhibition of the SecA component of the translocase in vitro, and hence, HSI molecules may target new unknown components that directly or indirectly affect protein secretion. The results provided proof of the principle that the new broad HTS approach can yield attractive nanomolar inhibitors that have potential as new starting compounds for optimization to derive potential antibiotics.

Effect of Orento, a Traditional Japanese Medicine, on IL-6, IL-8 Secretion, Type 1 Collagen Production and Alkaline Phosphatase Secretion in the Human Osteosarcoma Cell Line Saos-2.

Background: Orento, a traditional Japanese medicine, is known as Kampo medicine in Japan. We investigated the possible efficacy of Kampo medicine for periodontal disease. In this study, we examined the in vitro effects of orento on the proliferation of the inflammatory cytokines interleukin (IL)-6 and IL-8, the production of type 1 collagen, and the secretion of alkaline phosphatase (ALP) in the human osteosarcoma cell line Saos-2 (Saos-2 cells). Methods: The proliferation of Saos-2 cells was assessed by MTT assay. IL-6 and IL-8 levels, type 1 collagen production and ALP secretion were evaluated using enzyme-linked immunosorbent assay and ALP assays. Saos-2 cells were treated with or without 0.1, 1, 10, 100 and 1000 μg/mL of orento for 24 h. Results: Orento (10 μg/mL) significantly induced the proliferation of Saos-2 cells. At this concentration, orento suppressed IL-6 and IL-8 and enhanced type 1 collagen production and ALP secretion. Conclusions: These results indicate that orento controls the IL-6 and IL-8 secretion and cellular metabolism of osteoblasts, resulting in the secretion of early bone-related biomarkers.

Cathepsin K regulates localization and secretion of Tartrate-Resistant Acid Phosphatase (TRAP) in TRAP-overexpressing MDA-MB-231 breast cancer cells

BackgroundTartrate–resistant acid phosphatase (TRAP/ ACP5) belongs to the binuclear metallophosphatase family and is present in two isoforms. The primary translation product is an uncleaved TRAP 5a isoform with low phosphatase activity. TRAP 5a can be post-translationally processed to a cleaved TRAP 5b isoform with high phosphatase activity by e.g. cysteine proteinases, such as Cathepsin K (CtsK). The relevance of the phosphatase activity of TRAP 5b has been demonstrated for proliferation, migration and invasion of cancer cells. TRAP-overexpressing MDA-MB-231 breast cancer cells displayed higher levels of TRAP 5a and efficient processing of TRAP 5a to TRAP 5b protein, but no changes in levels of CtsK when compared to mock-transfected cells. In TRAP-overexpressing cells colocalization of TRAP 5a and proCtsK was augmented, providing a plausible mechanism for generation of TRAP 5b. CtsK expression has been associated with cancer progression and has been pharmacologically targeted in several clinical studies.ResultsIn the current study, CtsK inhibition with MK-0822/Odanacatib did not abrogate the formation of TRAP 5b, but reversibly increased the intracellular levels of a N-terminal fragment of TRAP 5b and reduced secretion of TRAP 5a reversibly. However, MK-0822 treatment neither altered intracellular TRAP activity nor TRAP-dependent cell migration, suggesting involvement of additional proteases in proteolytic processing of TRAP 5a. Notwithstanding, CtsK was shown to be colocalized with TRAP and to be involved in the regulation of secretion of TRAP 5a in a breast cancer cell line, while it still was not essential for processing of TRAP 5a to TRAP 5b isoform.ConclusionIn cancer cells multiple proteases are involved in cleaving TRAP 5a to high-activity phosphatase TRAP 5b. However, CtsK-inhibiting treatment was able to reduce secretion TRAP 5a from TRAP-overexpressing cancer cells.

OsHAD1, a Haloacid Dehalogenase-Like APase, Enhances Phosphate Accumulation.

Phosphorus (P) deficiency limits plant growth and yield. Since plants can absorb only the inorganic form of P (Pi), a large portion of soil P (organic and inorganic P complexes) remains unused. Here, we identified and characterized a PHR2-regulated, novel low-Pi-responsive haloacid dehalogenase (HAD)-like hydrolase, OsHAD1 While OsHAD1 is a functional HAD protein having both acid phosphatase and phytase activities, it showed little homology with other known low-Pi-responsive HAD superfamily members. Recombinant OsHAD1 is active at acidic pH and dephosphorylates a broad range of organic and inorganic P-containing substrates, including phosphorylated serine and sodium phytate. Exogenous application of recombinant OsHAD1 protein in growth medium supplemented with phytate led to marked increases in growth and total P content of Pi-deficient wild-type rice (Oryza sativa) seedlings. Furthermore, overexpression of OsHAD1 in rice resulted in enhanced phosphatase activity, biomass, and total and soluble P contents in Pi-deficient transgenic seedlings treated with phytate as a restricted Pi source. Gene expression and metabolite profiling revealed enhanced Pi starvation responses, such as up-regulation of multiple genes involved in Pi uptake and solubilization, accumulation of organic acids, enhanced secretory phosphatase activity, and depletion of ATP in overexpression lines as compared with the wild type. To elucidate the underlying regulatory mechanisms of OsHAD1, we performed in vitro pull-down assays, which revealed the association of OsHAD1 with protein kinases such as OsNDPKs. We conclude that, besides dephosphorylation of cellular organic P, OsHAD1 in coordination with kinases may regulate the phosphorylation status of downstream targets to accomplish Pi homeostasis under limited Pi supply.

Mutations improving production and secretion of extracellular lipase by Burkholderia glumae PG1.

Burkholderia glumae is a Gram-negative phytopathogenic bacterium known as the causative agent of rice panicle blight. Strain B. glumae PG1 is used for the production of a biotechnologically relevant lipase, which is secreted into the culture supernatant via a type II secretion pathway. We have comparatively analyzed the genome sequences of B. glumae PG1 wild type and a lipase overproducing strain obtained by classical strain mutagenesis. Among a total number of 72 single nucleotide polymorphisms (SNPs) identified in the genome of the production strain, two were localized in front of the lipAB operon and were analyzed in detail. Both mutations contribute to a 100-fold overproduction of extracellular lipase in B. glumae PG1 by affecting transcription of the lipAB operon and efficiency of lipase secretion. We analyzed each of the two SNPs separately and observed a stronger influence of the promoter mutation than of the signal peptide modification but also a cumulative effect of both mutations. Furthermore, fusion of the mutated LipA signal peptide resulted in a 2-fold increase in secretion of the heterologous reporter alkaline phosphatase from Escherichia coli.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-015-7041-z) contains supplementary material, which is available to authorized users.

Control of Acid Phosphatases Expression from Aspergillus niger by Soil Characteristics

This work studied the acid phosphatase (APase) activity from culture medium (extracellular, eAPase) and mycelial extract (intracellular, iAPase) ofAspergillus niger F111. The influence of fungus growth and phosphate concentration of the media on the synthesis and secretion of phosphatase was demonstrated. The effects of pH, substrate concentration and inorganic and organic compounds added to the reaction mixture on APase activity were also studied. Both enzymes were repressed by high concentrations of phosphate. Overexpression of iAPase in relation to eAPase was detected; iAPase activity was 46.1 times higher than eAPase. The maximal activity of eAPase was after 24h of fungus growth and for iAPase was after 96h. Optimal pH and substrate concentrations were 4.5 and 8.0 mM, respectively. Michaelis-Menten constant (Km) for the hydrolysis of p-nitrophenyl phosphate was 0.57 mM with Vmax = 14,285.71 U mg-1 mycelium for the iAPase and 0.31 mM with V max = 147.06 U mg-1 mycelium for eAPase. Organic substances had little effect on acid phosphatases when compared with the salts. Both the APases were inhibited by 10 mM KH 2PO4 and 5 mM (NH4)2MoO4; eAPase was also inhibited by 1 mM CoCl2.

Systematic Analysis of Mycobacterial Acylation Reveals First Example of Acylation-mediated Regulation of Enzyme Activity of a Bacterial Phosphatase

Protein lysine acetylation is known to regulate multiple aspects of bacterial metabolism. However, its presence in mycobacterial signal transduction and virulence-associated proteins has not been studied. In this study, analysis of mycobacterial proteins from different cellular fractions indicated dynamic and widespread occurrence of lysine acetylation. Mycobacterium tuberculosis proteins regulating diverse physiological processes were then selected and expressed in the surrogate host Mycobacterium smegmatis. The purified proteins were analyzed for the presence of lysine acetylation, leading to the identification of 24 acetylated proteins. In addition, novel lysine succinylation and propionylation events were found to co-occur with acetylation on several proteins. Protein-tyrosine phosphatase B (PtpB), a secretory phosphatase that regulates phosphorylation of host proteins and plays a critical role in Mycobacterium infection, is modified by acetylation and succinylation at Lys-224. This residue is situated in a lid region that covers the enzyme's active site. Consequently, acetylation and succinylation negatively regulate the activity of PtpB.

Protein Phosphatases of Pathogenic Bacteria: Role in Physiology and Virulence.

The role of protein phosphatases in pathogenic bacteria has been studied extensively over the last two decades. Ser/Thr and Tyr phosphatases are associated with growth and virulence of many bacteria. These phosphatases control kinase-mediated functions and return the proteins to their unmodified state. Biochemical, structural, and functional studies, in addition to extensive genetic characterization, have highlighted the importance of phosphatases in bacteria. However, questions remain regarding the mechanisms driving localization of secretory phosphatases to cellular compartments, identification of receptor phosphatase sensory signals, and a possible role of cofactors and ligands in their functions. This review focuses on the role of Ser/Thr- and Tyr-specific phosphatases present in pathogenic bacteria, with an emphasis on the regulation of basic cellular processes and virulence. Furthermore, we highlight their clinical importance and analyze the development of drugs targeting protein phosphatases.

Pericellular Hydrogel/Nanonets Inhibit Cancer Cells

AbstractFibrils formed by proteins are vital components for cells. However, selective formation of xenogenous nanofibrils of small molecules on mammalian cells has yet to be observed. Here we report an unexpected observation of hydrogel/nanonets of a small D‐peptide derivative in pericellular space. Surface and secretory phosphatases dephosphorylate a precursor of a hydrogelator to trigger the self‐assembly of the hydrogelator and to result in pericellular hydrogel/nanonets selectively around the cancer cells that overexpress phosphatases. Cell‐based assays confirm that the pericellular hydrogel/nanonets block cellular mass exchange to induce apoptosis of cancer cells, including multidrug‐resistance (MDR) cancer cells, MES‐SA/Dx5. Pericellular hydrogel/nanonets of small molecules to exhibit distinct functions illustrates a fundamentally new way to engineer molecular assemblies spatiotemporally in cellular microenvironment for inhibiting cancer cell growth and even metastasis.

Pericellular hydrogel/nanonets inhibit cancer cells.

Fibrils formed by proteins are vital components for cells. However, selective formation of xenogenous nanofibrils of small molecules on mammalian cells has yet to be observed. Here we report an unexpected observation of hydrogel/nanonets of a small D-peptide derivative in pericellular space. Surface and secretory phosphatases dephosphorylate a precursor of a hydrogelator to trigger the self-assembly of the hydrogelator and to result in pericellular hydrogel/nanonets selectively around the cancer cells that overexpress phosphatases. Cell-based assays confirm that the pericellular hydrogel/nanonets block cellular mass exchange to induce apoptosis of cancer cells, including multidrug-resistance (MDR) cancer cells, MES-SA/Dx5. Pericellular hydrogel/nanonets of small molecules to exhibit distinct functions illustrates a fundamentally new way to engineer molecular assemblies spatiotemporally in cellular microenvironment for inhibiting cancer cell growth and even metastasis.

APPLICATION OF DENDRIMER/PLASMID hBMP-2 COMPLEXES LOADED INTO β-TCP/COLLAGEN SCAFFOLD IN THE TREATMENT OF FEMORAL DEFECTS IN RATS

Purpose: Plasmid loading into scaffolds to enhance sustained release of growth factors is an important focus of regenerative medicine. The aim of this study was to build gene-activated matrices (GAMs) and examine the bone augmentation properties. Methods: Generation 5 polyamidoamine dendrimers (G5 dPAMAM)/plasmid recombinant human bone morphogenetic protein-2 (rhBMP-2) complexes were immobilized into beta-tricalcium phosphate (β-TCP)/type I collagen porous scaffolds. After cultured with rat mesenchymal stem cells (rMSCs), transfection efficiencies were examined. The secretion of rhBMP-2 and alkaline phosphatase (ALP) were detected to evaluate the osteogenic properties. Scanning electron microscopy (SEM) was used to observe attachment and proliferation. Moreover, we applied these GAMs directly into freshly created segmental bone defects in rat femurs, and their osteogenic efficiencies were evaluated. Results: Released plasmid complexes were transfected into stem cells and were expressed, which caused osteogenic differentiations of rat mesenchymal stem cells (rMSCs). SEM analysis showed excellent cell attachment. Bioactivity of plasmid rhBMP-2 was maintained in vivo, and the X-ray observation, histological analysis and immunohistochemistry (IHC) of bone tissue demonstrated that the bone healing in segmental femoral defects was enhanced by implantation of GAMs. Conclusions: Such biomaterials offer therapeutic opportunities in critical-sized bone defects.

Production and secretion of a multifunctional ß-glucosidase by Humicola grisea var. thermoidea: effects of L-sorbose

The effects of L-sorbose on growth, morphology and production of a multifunctional s-glucosidase by the thermophilic fungus Humicola grisea var. thermoidea were investigated. Sorbose increased the lag phase period 3-fold and drastically altered the morphology of the fungal hyphae. Cellobiose and lactose were good inducers of the enzyme. The addition of 5 % sorbose to cultures containing 1 % cellobiose enhanced the extracellular levels of the s-glucosidase 3.3-fold with constant cytosolic and cell-wall bound levels, demonstrating stimulation of both enzyme synthesis and secretion. The stimulation of enzyme production by sorbose was dependent on the presence of cellobiose as inducer, since 2- to 3-fold inhibition was observed in lactose and glucose. Production and secretion of phosphatases and endoglucanases was not stimulated by sorbose, which did not affect the subcellular distribution of the s-glucosidase also. However, it reduced the uptake rates of glucose and cellobiose. Taken together, the results discarded increased non-specific enzyme secretion and/or increased release of the enzyme from the cell-wall as possible molecular mechanisms for the effects of sorbose on the production of the multifunctional s-glucosidase by H. grisea. An alternative mechanism, based on a prolonged action of cellobiose as inducer associated with a decreased catabolic repression by glucose, was discussed.

Secretory Phosphatases Deficient Mutant of Mycobacterium tuberculosis Imparts Protection at the Primary Site of Infection in Guinea Pigs

BackgroundThe failure of Mycobacterium bovis Bacille Calmette-Guérin to impart satisfactory protection against adult pulmonary tuberculosis has necessitated the development of more effective TB vaccines. The assumption that the vaccine strain should be antigenically as similar as possible to the disease causing pathogen has led to the evaluation of M.tuberculosis mutants as candidate tuberculosis vaccines.Methods/Principal FindingsIn this study, we have generated a mutant of M.tuberculosis (Mtb∆mms) by disrupting 3 virulence genes encoding a mycobacterial secretory acid phosphatase (sapM) and two phosphotyrosine protein phosphatases (mptpA and mptpB) and have evaluated its protective efficacy in guinea pigs. We observed that Mtb∆mms was highly attenuated in THP-1 macrophages. Moreover, no bacilli were recovered from the lungs and spleens of guinea pigs after 10 weeks of Mtb∆mms inoculation, although, initially, the mutant exhibited some growth in the spleens. Subsequently, when Mtb∆mms was evaluated for its protective efficacy, we observed that similar to BCG vaccination, Mtb∆mms exhibited a significantly reduced CFU in the lungs of guinea pigs when compared with the unvaccinated animals at 4 weeks after challenge. In addition, our observations at 12 weeks post challenge demonstrated that Mtb∆mms exhibited a more sustainable and superior protection in lungs as compared to BCG. However, the mutant failed to control the hematogenous spread as the splenic bacillary load between Mtb∆mms vaccinated and sham immunized animals was not significantly different. The gross pathological observations and histopathological observations corroborated the bacterial findings. Inspite of disruption of phosphatase genes in MtbΔmms, the lipid profiles of M.tuberculosis and MtbΔmms were identical indicating thereby that the phenotype of the mutant was ascribed to the loss of phosphatase genes and the influence was not related to any alteration in the lipid composition.Conclusions/SignificanceThis study highlights the importance of M.tuberculosis mutants in imparting protection against pulmonary TB.

The type II secretion system (Xcp) of Pseudomonas putida is active and involved in the secretion of phosphatases

The genome of the Gram-negative bacterium Pseudomonas putida harbours a complete set of xcp genes for a type II protein secretion system (T2SS). This study shows that expression of these genes is induced under inorganic phosphate (Pi ) limitation and that the system enables the utilization of various organic phosphate sources. A phosphatase of the PhoX family, previously designated UxpB, was identified, which was produced under low Pi conditions and transported across the cell envelope in an Xcp-dependent manner demonstrating that the xcp genes encode an active T2SS. The signal sequence of UxpB contains a twin-arginine translocation (Tat) motif as well as a lipobox, and both processing by leader peptidase II and Tat dependency were experimentally confirmed. Two different tat gene clusters were detected in the P. putida genome, of which one, named tat-1, is located adjacent to the uxpB and xcp genes. Both Tat systems appeared to be capable of transporting the UxpB protein. However, expression of the tat-1 genes was strongly induced by low Pi levels, indicating a function of this system in survival during Pi starvation.

Screening, evaluation and selection ofphosphate-solubilising fungi as potential biofertiliser

Phosphate-solubilising saprophytic fungi have a potential application in plant nutri- tion; therefore, the aim of this study was 1) to perform a screening and isolation of native phosphofungi from volcanic soils of southern Chile, 2) to select a liquid medium for the evaluation of these phosphofungi and 3) to test a selected phospho- fungus as a biofertiliser in a volcanic soil. The phosphofungi were screened using Martin medium (rose bengal-streptomycin agar) with calcium phosphate (Ca 3 (PO 4 ) 2 ) or calcium phytate as the phosphorus source. Six promising strains (Penicillium sp., Penicillium albidum, Penicillium thomii, Penicillium restrictum, Penicillium frequentans and Gliocladium roseum) were evaluated in the liquid media of Agnihotri, Asea-Wakelin, Pikovskaya and Na- has. The soluble phosphorus, acid phosphatase activity, pH and fungal biomass were determined. In most soils, the greatest proportion of phosphofungi solubilised organic P. The Asea-Wakelin medium appears to be the medium of choice for the quantitative evaluation of phosphofungi isolated from the volcanic soils tested. Penicillium al- bidum was selected as a potential biofertiliser due to its capacity to solubilise both inorganic and organic P via its specific solubilising activity (64 mg P/g fungus), phosphatase secretion and enhancement of the growth and mineral nutrition of let- tuce plants growing in a volcanic soil.

Enhancement of acid phosphatase secretion and Pi acquisition inSuaeda fruticosaon calcareous soil by high saline level

The aim of this study was to identify the relationship between the adaptive processes of Suaeda fruticosa for Pi acquisition and the physic-chemical and biological characteristics of two soil types under moderate and high saline conditions. Four treatments were established in pots: namely SS100, SS600, CS100 and CS600 where SS stood for sandy soil and CS for calcareous soil, and the indexes 100 and 600 were NaCl concentrations (mM) in irrigation distilled water. Assuming that Pi per g of plant biomass is an indicator of plant efficiency for P acquisition, the results showed that Pi acquisition was easiest on SS100 and was difficult on CS100. The differences in Pi acquisition between plants on SS100 and CS100 could be attributed to the low root surface area (-30%) and to the low alkaline phosphatases (Pases) activities (-50%) in calcareous rhizospheric soil. The high salinity level had no effect on the efficiency of P acquisition on SS but increased this parameter on CS (+50%). In the latter soil type, high acid phosphatase activities were observed in rhizospheric soil at high salinity level. Acid phosphatase seemed to be secreted from the roots. The higher secretion of acid phosphatase in this soil was related to the root lipid peroxidation in response to elevated salinity associated with the augmentation of unsaturated acids which might induce an oxidative damage of the root membrane. Thus we can conclude that in deficient soil such as calcareous, the efficiency of P acquisition in S. fruticosa which was difficult at moderate salinity level can be enhanced by high salinity level.

Bile secretion and palm oil diets in Wistar rats

PurposeChronic feeding with thermoxidized palm oil causes tissue damage. The purpose of this paper is to ascertain whether chronic feeding of oxidized and fresh palm oil affects biliary secretion.Design/methodology/approachAlbino Wistar rats were divided into three groups of ten rats each. The first group was the control and were fed on normal rat chow, while the second (FPO) and third group (TPO) were fed diet containing either fresh or thermoxidized palm oil (15per cent (w/w)) for 14 weeks. Biliary secretion, bilirubin, electrolytes, cholesterol and serum levels of alkaline phosphatase, aspartate aminotransferase and alanine aminotransferase enzymes were measured.FindingsBiliary secretion in TPO was significantly (p &lt; 0.05) lower compared with the control or FPO. Electrolytes (Na+, Cl− and HCO3−) content of bile were significantly (p &lt; 0.05) lower in TPO compared with control or FPO group. Conjugated and un‐conjugated bilirubin levels were significantly (p &lt; 0.05) elevated in TPO compared with control or FPO. The levels of serum liver enzymes were significantly (p &lt; 0.001) higher in TPO than control or FPO. The cholesterol level in the TPO was significantly higher (p &lt; 0.001) than in control or FPO.Research limitations/implicationsThe non‐clinical approach and use of experimental animals present limitations to the study.Originality/valueThese results suggest that long‐term feeding of thermally oxidized palm oil alters bile composition and decreases biliary flow due to its hazardous free radicals.

Abstract LB-375: Development of new compounds specifically targeting prostate cancer bone metastases

Abstract Introduction: Bone metastases are a major cause of morbidity for men with prostate cancer (PCa). Although PCa cells produce osteoblastic metastases, there is an initial and ongoing osteoclast (OC)-mediated osteolytic phase that is essential for PCa bone metastases. OC-mediated bone resorption is dependent upon OC secretion of bone acid phosphatase. PCa cells in bone secrete high levels of prostatic acid phosphatase (PAP) which differs from BAP in that PAP enzymatic activity is inhibited by tartrate and glyceric acid, whereas BAP is tartrate-resistant. Hypothesis: PAP secreted by human PCa cells in bone acts similarly to OC-derived bone acid phosphatase to degrade bone matrix and enhance PCa bone-targeting and growth in bone. We developed new compounds consisting of glyceric acid or tartrate (known PAP inhibitors) conjugated to a bisphosphonate to inhibit PCa bone metastases. Methods: PCa bone metastases derived from 7 patients were immunostained for androgen receptor (AR), prostate specific antigen (PSA) and PAP expression. The VCaP human PCa cell line derived from a vertebral metastases was inoculated intratibially into SCID mice (n=8) and bone lesions immunostained for AR, PSA, and PAP. Co-cultures of MC3T3 osteoblasts and VCaP cells were treated with tartrate and the effects on cell number, PAP and bone alkaline phosphatase (ALP) secretion measured by ELISA. Glyceric Acid and Tartaric acid were then conjugated with alendronate and the effects of these new conjugates on PAP enzymatic activity were compared to alendronate alone and tartrate alone. Results: Human PCa bone lesions (7/7) had strongly positive expression of PAP, with little AR or PSA expression. 8/8 SCID mice inoculated with VCaP cells intratibially developed osteoblastic VCaP lesions and these similarly had high PAP expression and no PSA expression. Tartrate addition to co-cultures of VCaP and MC3T3 osteoblasts reduced MC3T3 cell numbers and ALP secretion. In vitro testing of the conjugates revealed that the glyceric acid-alendronate conjugate inhibited PAP enzymatic activity but the tartrate-alendronate conjugate and alendronate alone had no significant inhibitory activity. Discussion: We have evidence that PAP secretion by PCa cells enhances their bone metastatic ability. We have developed new bone-targeting agents that consist of alendronate conjugated to PAP-inhibitory small molecules for further development as oral agents to prevent and treat PCa bone metastases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-375.