David J. Schultz

RNA isolation from recalcitrant plant tissue

The isolation of high-quality RNA from various tissues (leaves, pedicels, glandular trichomes) of garden geranium (Pelargonium xhortorum) using various published methods is difficult due to numerous oxidizing compounds. A new RNA extraction method was developed through the combination and modification of two separate procedures (Rochester et al., 1986; Manning 1991). In addition to geranium tissues, this method is successful when used with other recalcitrant tissues such as mature needles of white pine (Pinus strobus) and mature leaves of poinsettia (Euphorbia pulcherrima). RNA quality was judged by spectrophotometric readings, denaturing agarose gels, and successful reverse transcription.

Proteomic and functional characterisation of platelet microparticle size classes

UNLABELLED Activated platelets release large lipid-protein complexes termed microparticles. These platelet microparticles (PMP) are composed of vesicular fragments of the plasma membrane and alpha-granules. PMP facilitate coagulation, promote platelet and leukocyte adhesion to the subendothelial matrix, support angiogenesis and stimulate vascular smooth muscle proliferation. OBJECTIVES PMP were separated into 4 size classes to facilitate identification of active protein and lipid components. PMP were obtained from activated human platelets and separated into 4 size classes by gel filtration chromatography. Proteins were identified using 2-dimensional, liquid chromatography tandem mass spectrometry. Functional effects on platelets were determined using the PFA-100 and on endothelial cells by measuring transendothelial cell electrical resistance. PMP size classes differed significantly in their contents of plasma membrane receptors and adhesion molecules, chemokines, growth factors and protease inhibitors. The two smallest size classes (3 and 4) inhibited collagen/adenosine-diphosphate-mediated platelet thrombus formation, while fractions 2 and 4 stimulated barrier formation by endothelial cells. Heat denaturation blocked the effect of fraction 4 on endothelial cell function, but not fraction 2 implying that the active component in fraction 4 is a protein and in fraction 2 is a heat-stable protein or lipid but not sphingosine-1-phosphate. Proteomic and functional analysis of PMP size fractions has shown that PMP can be separated into different size classes that differ in protein components, protein/lipid ratio, and functional effects on platelets and endothelial cells. This analysis will facilitate identification of active components in the PMP and clarify their involvement in diseases such as atherosclerosis and cancer.

Anacardic acid inhibits estrogen receptor alpha-DNA binding and reduces target gene transcription and breast cancer cell proliferation

Anacardic acid (AnAc; 2-hydroxy-6-alkylbenzoic acid) is a dietary and medicinal phytochemical with established anticancer activity in cell and animal models. The mechanisms by which AnAc inhibits cancer cell proliferation remain undefined. AnAc 24:1ω5 was purified from geranium (Pelargonium × hortorum) and shown to inhibit the proliferation of estrogen receptor α (ERα)–positive MCF-7 and endocrine-resistant LCC9 and LY2 breast cancer cells with greater efficacy than ERα-negative primary human breast epithelial cells, MCF-10A normal breast epithelial cells, and MDA-MB-231 basal-like breast cancer cells. AnAc 24:1ω5 inhibited cell cycle progression and induced apoptosis in a cell-specific manner. AnAc 24:1ω5 inhibited estradiol (E2)–induced estrogen response element (ERE) reporter activity and transcription of the endogenous E2 target genes pS2, cyclin D1, and cathepsin D in MCF-7 cells. AnAc 24:1ω5 did not compete with E2 for ERα or ERβ binding, nor did AnAc 24:1ω5 reduce ERα or ERβ steady-state protein levels in MCF-7 cells; rather, AnAc 24:1ω5 inhibited ER-ERE binding in vitro. Virtual screening with the molecular docking software Surflex evaluated AnAc 24:1ω5 interaction with ERα ligand binding (LBD) and DNA binding (DBD) domains in conjunction with experimental validation. Molecular modeling revealed AnAc 24:1ω5 interaction with the ERα DBD but not the LBD. Chromatin immunoprecipitation experiments revealed that AnAc 24:1ω5 inhibited E2-ERα interaction with the endogenous pS2 gene promoter region containing an ERE. These data indicate that AnAc 24:1ω5 inhibits cell proliferation, cell cycle progression, and apoptosis in an ER-dependent manner by reducing ER-DNA interaction and inhibiting ER-mediated transcriptional responses. Mol Cancer Ther; 9(3); 594–605

Chemoprevention of Rat Mammary Carcinogenesis by Apiaceae Spices

Scientific evidence suggests that many herbs and spices have medicinal properties that alleviate symptoms or prevent disease. In this study, we examined the chemopreventive effects of the Apiaceae spices, anise, caraway, and celery seeds against 17β-estrogen (E2)-mediated mammary tumorigenesis in an ACI (August-Copenhagen Irish) rat model. Female ACI rats were given either control diet (AIN 93M) or diet supplemented with 7.5% (w/w) of anise, caraway, or celery seed powder. Two weeks later, one half of the animals in each group received subcutaneous silastic implants of E2. Diet intake and body weight were recorded weekly, and animals were euthanized after 3 and 12 weeks. E2-treatment showed significantly (2.1- and 3.4-fold) enhanced growth of pituitary gland at 3 and 12 weeks, respectively. All test spices significantly offset the pituitary growth by 12 weeks, except celery which was effective as early as three weeks. Immunohistochemical analysis for proliferative cell nuclear antigen (PCNA) in mammary tissues showed significant reduction in E2-mediated mammary cell proliferation. Test spices reduced the circulating levels of both E2 and prolactin at three weeks. This protection was more pronounced at 12 weeks, with celery eliciting the highest effect. RT-PCR and western blot analysis were performed to determine the potential molecular targets of the spices. Anise and caraway diets significantly offset estrogen-mediated overexpression of both cyclin D1 and estrogen receptor α (ERα). The effect of anise was modest. Likewise, expression of CYP1B1 and CYP1A1 was inhibited by all test spices. Based on short-term molecular markers, caraway was selected over other spices based on its enhanced effect on estrogen-associated pathway. Therefore, a tumor-end point study in ACI rats was conducted with dietary caraway. Tumor palpation from 12 weeks onwards revealed tumor latency of 29 days in caraway-treated animals compared with first tumor appearance at 92 days in control group. At the end of the study (25 weeks), the tumor incidence was 96% in the control group compared with only 70% in the caraway group. A significant reduction in tumor volume (661 ± 123 vs. 313 ± 81 mm3) and tumor multiplicity (4.2 ± 0.4 vs. 2.5 ± 0.5 tumors/animal) was also observed in the caraway group compared with the control group. Together, our data show dietary caraway can significantly delay and prevent the hormonal mammary tumorigenesis by modulating different cellular and molecular targets.

Anacardic Acid, Salicylic Acid, and Oleic Acid Differentially Alter Cellular Bioenergetic Function in Breast Cancer Cells.

Anacardic acid is a dietary and medicinal phytochemical that inhibits breast cancer cell proliferation and uncouples oxidative phosphorylation (OXPHOS) in isolated rat liver mitochondria. Since mitochondrial‐targeted anticancer therapy (mitocans) may be useful in breast cancer, we examined the effect of anacardic acid on cellular bioenergetics and OXPHOS pathway proteins in breast cancer cells modeling progression to endocrine‐independence: MCF‐7 estrogen receptor α (ERα)+ endocrine‐sensitive; LCC9 and LY2 ERα+, endocrine‐resistant, and MDA‐MB‐231 triple negative breast cancer (TNBC) cells. At concentrations similar to cell proliferation IC50s, anacardic acid reduced ATP‐linked oxygen consumption rate (OCR), mitochondrial reserve capacity, and coupling efficiency while increasing proton leak, reflecting mitochondrial toxicity which was greater in MCF‐7 compared to endocrine‐resistant and TNBC cells. These results suggest tolerance in endocrine‐resistant and TNBC cells to mitochondrial stress induced by anacardic acid. Since anacardic acid is an alkylated 2‐hydroxybenzoic acid, the effects of salicylic acid (SA, 2‐hydroxybenzoic acid moiety) and oleic acid (OA, monounsaturated alkyl moiety) were tested. SA inhibited whereas OA stimulated cell viability. In contrast to stimulation of basal OCR by anacardic acid (uncoupling effect), neither SA nor OA altered basal OCR‐ except OA inhibited basal and ATP‐linked OCR, and increased ECAR, in MDA‐MB‐231 cells. Changes in OXPHOS proteins correlated with changes in OCR. Overall, neither the 2‐hydroxybenzoic acid moiety nor the monounsaturated alky moiety of anacardic acid is solely responsible for the observed mitochondria‐targeted anticancer activity in breast cancer cells and hence both moieties are required in the same molecule for the observed effects. J. Cell. Biochem. 117: 2521–2532, 2016.

Ustilago maydis phosphodiesterases play a role in the dimorphic switch and in pathogenicity

Components of the cAMP (cyclic AMP) signalling cascades are conserved from fungi to humans, and are particularly important for fungal dimorphism and pathogenicity. Previous work has described two phosphodiesterases, UmPde1 and UmPde2, in Ustilago maydis which show strong phosphodiesterase activity. We further characterized the biological function(s) of these phosphodiesterases in U. maydis. Specifically, we examined their possible role(s) in regulation of the cAMP-dependent protein kinase A (PKA) pathway and their roles in filamentous growth and pathogenicity. We found that UmPde1, which shares 35 % similarity with Cryptococcus neoformans Pde1, also displays functional homology with this enzyme. UmPde1 complements the capsule-formation defect of C. neoformans strains deleted for Pde1. In U. maydis, the cell morphology of the umpde1 deletion mutant resembled the multiple budding phenotypes seen with the ubc1 mutant, which lacks the regulatory subunit of PKA. Interestingly, on low-ammonium medium, umpde2 deletion strains showed a reduction in filamentation that was comparable to that of ubc1 deletion strains; however, umpde1 deletion strains showed normal filamentation on low-ammonium medium. Furthermore, both the ubc1 deletion strain in which the PKA pathway was constitutively active and the umpde1 deletion strains were significantly reduced in pathogenicity, while the umpde2 deletion strains showed a trend for reduced pathogenicity compared with wild-type strains. These data support a role for the phosphodiesterases UmPde1 and UmPde2 in regulating the U. maydis cAMP-dependent PKA pathway through modulation of cAMP levels, thus affecting dimorphic growth and pathogenicity.

Two Phosphodiesterases from Ustilago Maydis Share Structural and Biochemical Properties with Non-Fungal Phosphodiesterases

The dependence of Protein Kinase A (PKA) activity on cAMP levels is an important facet of the dimorphic switch between budding and filamentous growth as well as for pathogenicity in some fungi. To better understand these processes in the pathogenic fungus Ustilago maydis, we characterized the structure and biochemical functions of two phosphodiesterase (PDE) genes. Phosphodiesterases are enzymes involved in cAMP turnover and thus, contribute to the regulation of the cAMP-PKA signaling pathway. Two predicted homologs of PDEs were identified in the genome of U. maydis and hypothesized to be involved in cAMP turnover, thus regulating activity of the PKA catalytic subunit. Both umpde1 and umpde2 genes contain domains associated with phosphodiesterase activity predicted by InterPro analysis. Biochemical characterization of recombinantly produced UmPde1 (U. maydis Phosphodiesterase I) and UmPde2 demonstrated that both enzymes have phosphodiesterase activity in vitro, yet neither was inhibited by the phosphodiesterase inhibitor IBMX. Moreover, UmPde1 is specific for cAMP, while UmPde2 has broader substrate specificity, utilizing cAMP and cGMP as substrates. In addition, UmPde2 was also found to have nucleotide phosphatase activity that was higher with GMP compared to AMP. These results demonstrate that UmPde1 is a bona fide phosphodiesterase, while UmPde2 has more general activity as a cyclic nucleotide phosphodiesterase and/or GMP/AMP phosphatase. Thus, UmPde1 and UmPde2 likely have important roles in cell morphology and development and share some characteristics with a variety of non-fungal phosphodiesterases.

Chapter Six – Anacardic Acid Biosynthesis and Bioactivity

This chapter focuses on the biosynthesis of anacardic acid with an emphasis on the type III polyketide synthase family as well as the latest work understanding lipid metabolism in geranium glandular trichomes. The chapter also reviews the bioactivity of anacardic acid with an emphasis on utilization of anacardic acids for agricultural and medicinal applications. Anacardic acids are found in a number of plants, most commonly within the Anacardiaceae family. Based on results of labeling studies, anacardic acids have been proposed to be synthesized from fatty acids by action of a type III polyketide synthase adding acetate units from malonyl-CoA. Utilizing isolated geranium glandular trichome cells, a fatty acid CoA ester, as well as malonyl-CoA and acetate, have been shown to be incorporated into anacardic acids, verifying the proposed polyketide synthesis pathway.

Genome-wide miRNA response to anacardic acid in breast cancer cells

MicroRNAs are biomarkers and potential therapeutic targets for breast cancer. Anacardic acid (AnAc) is a dietary phenolic lipid that inhibits both MCF-7 estrogen receptor α (ERα) positive and MDA-MB-231 triple negative breast cancer (TNBC) cell proliferation with IC50s of 13.5 and 35 μM, respectively. To identify potential mediators of AnAc action in breast cancer, we profiled the genome-wide microRNA transcriptome (microRNAome) in these two cell lines altered by the AnAc 24:1n5 congener. Whole genome expression profiling (RNA-seq) and subsequent network analysis in MetaCore Gene Ontology (GO) algorithm was used to characterize the biological pathways altered by AnAc. In MCF-7 cells, 69 AnAc-responsive miRNAs were identified, e.g., increased let-7a and reduced miR-584. Fewer, i.e., 37 AnAc-responsive miRNAs were identified in MDA-MB-231 cells, e.g., decreased miR-23b and increased miR-1257. Only two miRNAs were increased by AnAc in both cell lines: miR-612 and miR-20b; however, opposite miRNA arm preference was noted: miR-20b-3p and miR-20b-5p were upregulated in MCF-7 and MDA-MB-231, respectively. miR-20b-5p target EFNB2 transcript levels were reduced by AnAc in MDA-MB-231 cells. AnAc reduced miR-378g that targets VIM (vimentin) and VIM mRNA transcript expression was increased in AnAc-treated MCF-7 cells, suggesting a reciprocal relationship. The top three enriched GO terms for AnAc-treated MCF-7 cells were B cell receptor signaling pathway and ribosomal large subunit biogenesis and S-adenosylmethionine metabolic process for AnAc-treated MDA-MB-231 cells. The pathways modulated by these AnAc-regulated miRNAs suggest that key nodal molecules, e.g., Cyclin D1, MYC, c-FOS, PPARγ, and SIN3, are targets of AnAc activity.

Transcriptional analysis of mating and pre-infection stages of the anther smut, Microbotryum lychnidis-dioicae

Microbotryum lychnidis-dioicae is an obligate biotrophic parasite of the wildflower species, Silene latifolia. This dikaryotic fungus, commonly known as an anther smut, requires that haploid, yeast-like sporidia of opposite mating types fuse and differentiate into dikaryotic hyphae that penetrate host tissue as part of the fungal lifecycle. Mating occurs under conditions of cool temperatures and limited nutrients. Further development requires host cues or chemical mimics, including a variety of lipids, e.g., phytols. To identify global changes in transcription associated with developmental shifts, RNA-Seq was conducted at several in vitro stages of fungal propagation, i.e., haploid cells grown independently on rich and nutrient-limited media, mated cells on nutrient-limited media, as well as a time-course of such mated cells exposed to phytol. Comparison of haploid cells grown under rich and nutrient-limited conditions identified classes of genes likely associated with general nutrient availability, including components of the RNAi machinery. Some gene enrichment patterns comparing the nutrient-limited and mated transcriptomes suggested gene expression changes associated with the mating program (e.g., homeodomain binding proteins, secreted proteins, proteins unique to M. lychnidis-dioicae¸ multicopper oxidases, and RhoGEFs). Analysis for phytol treatment compared with mated cells alone allowed identification of genes likely involved in the dikaryotic switch (e.g., oligopeptide transporters). Gene categories of particular note in all three conditions included those in the major facilitator superfamily, proteins containing PFAM domains of the secretory lipase family, as well as proteins predicted to be secreted, many of which have the hallmarks of fungal effectors with potential roles in pathogenicity.