Why Leaves Turn Red in Autumn. The Role of Anthocyanins in Senescing Leaves of Red-Osier Dogwood

Why the leaves of many woody species accumulate anthocyanins prior to being shed has long puzzled biologists because it is unclear what effects anthocyanins may have on leaf function. Here, we provide evidence for red-osier dogwood (Cornus stolonifera) that anthocyanins form a pigment layer in the palisade mesophyll layer that decreases light capture by chloroplasts. Measurements of leaf absorbance demonstrated that red-senescing leaves absorbed more light of blue-green to orange wavelengths (495–644 nm) compared with yellow-senescing leaves. Using chlorophyll a fluorescence measurements, we observed that maximum photosystem II (PSII) photon yield of red-senescing leaves recovered from a high-light stress treatment, whereas yellow-senescing leaves failed to recover after 6 h of dark adaptation, which suggests photo-oxidative damage. Because no differences were observed in light response curves of effective PSII photon yield for red- and yellow-senescing leaves, differences between red- and yellow-senescing cannot be explained by differences in the capacities for photochemical and non-photochemical light energy dissipation. A role of anthocyanins as screening pigments was explored further by measuring the responses PSII photon yield to blue light, which is preferentially absorbed by anthocyanins, versus red light, which is poorly absorbed. We found that dark-adapted PSII photon yield of red-senescing leaves recovered rapidly following illumination with blue light. However, red light induced a similar, prolonged decrease in PSII photon yield in both red- and yellow-senescing leaves. We suggest that optical masking of chlorophyll by anthocyanins reduces risk of photo-oxidative damage to leaf cells as they senesce, which otherwise may lower the efficiency of nutrient retrieval from senescing autumn leaves.

Biological activities of compounds from Piper species

It is well established that secondary metabolites play an important role in plant chemical defense. In an effort to find natural herbicides research on plant growth regulatory activity of secondary metabolites has received more and more attention recently. The genus Piper has been an important source for useful secondary metabolites.^ Crude extracts from Piper species inhibited gram-positive bacteria and higher plant growth under laboratory conditions. Bioassay-guided isolation and purification lead to the identification of safrole, a phenylpropene, as the responsible agent for the inhibitory activity. Safrole was found to leach from naturally fallen leaves with water. Mechanisms of plant growth inhibition by safrole were investigated. Disassociation of cell membrane from cell walls was determined to be a major cause.^ Phenylpropenes structurally similar to safrole had similar phytogrowth inhibitory activity. It is postulated that phenylpropanoids are an important group of naturally occurring secondary metabolites in plant-plant interactions. ^

Interactions of different arsenic species with thols: Chemical and biological implications

Arsenic has been classified as a group I carcinogen. It has been ranked number one in the CERCLA priority list of hazardous substances due to its frequency, toxicity and potential for human exposure. Paradoxically, arsenic has been employed as a successful chemotherapeutic agent for acute promyelocytic leukemia and has found some success in multiple myeloma. Since arsenic toxicity and efficacy is species dependent, a speciation method, based on the complementary use of reverse phase and cation exchange chromatography, was developed. Inductively coupled plasma mass spectrometer (ICP-MS), as an element specific detector, and electrospray ionization mass spectrometer (ESI-MS), as a molecule specific detector, were employed. Low detection limits in the µg. L−1 range on the ICP-MS and mg. L−1 range on the ESI-MS were obtained. The developed methods were validated against each other through the use of a Deming plot. With the developed speciation method, the effects of both pH on the stability of As species and reduced glutathione (GSH) concentration on the formation and stability of arsenic glutathione complexes were studied. To identify arsenicals in multiple myeloma (MM) cell lines post arsenic trioxide (ATO) and darinaparsin (DAR) incubation, an extraction method based on the use of ultrasonic probe was developed. Extraction tools and solvents were evaluated and the effect of GSH concentration on the quantitation of arsenic glutathione (As-GSH) complexes in MM cell extracts was studied. The developed method was employed for the identification of metabolites in DAR incubated cell lines where the effect of extraction pH, DAR incubation concentration and incubation time on the relative distribution of the As metabolites was assessed. A new arsenic species, dimethyarsinothioyl glutathione (DMMTA V-GS), a pentavalent thiolated arsenical, was identified in the cell extracts through the use of liquid chromatography tandem mass spectrometry. The formation of the new metabolite in the extracts was dependent on the decomposition of s-dimethylarsino glutathione (DMA(GS)). These results have major implications in both the medical and toxicological fields of As because they involve the metabolism of a chemotherapeutic agent and the role sulfur compounds play in this mechanism.

Role of Pseudomonas aeruginosa mifSR Two-Component System in Antibiotic Resistance and Biofilm Formation

Pseudomonas aeruginosa is an opportunistic pathogen found in a wide variety of environments. It is one of the leading causes of morbidity and mortality in cystic fibrosis patients, and one of the main sources of nosocomial infections in the United States. One of the most prominent features of this pathogen is its wide resistance to antibiotics. P. aeruginosa employs a variety of mechanisms including efflux pumps and the expression of B-lactamases to overcome antibiotic treatment. Two chromosomally encoded lactamases, ampC and poxB, have been identified in P. aeruginosa. Sequence analyses have shown the presence of a two-component system (TCS) called MifSR (MifS-Sensor and MifR-Response Regulator), immediately upstream of the poxAB operon. It is hypothesized that the MifSR TCS is involved in B-lactam resistance via the regulation of poxB. Recently, the response regulator MifR has been reported to play a crucial role in biofilm formation, a major characteristic of chronic infections and increased antibiotic resistance. In this study, mifR and mifSR deletion mutants were constructed, and compared to the wild type parent strain PAOl for differences in growth and B-lactam sensitivity. Results obtained thus far indicate that mifR and mifSR are not essential for growth, and do not confer B-lactam resistance under the conditions tested. This study is significant because biofilm formation and antibiotic resistance are two hallmarks of P. aeruginosa infections, and finding a link between these two may lead to the development of improved treatment strategies.

Role of Pseudomonas aeruginosa PA4157in Quorum Sensing and Iron Transport Regulation

Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen, is a pnmary contributing factor responsible for the morbidity and mortality in patients with cystic fibrosis. One of the trademarks of P. aeruginosa is its ability to resist antibiotics. P. aeruginosa does so in part through the LysR-type transcription factor, AmpR. To identify additional members of the AmpR regulon, a new algorithm called iterative enhancement of motifs was used to identify putative AmpR binding sites upstream of open reading frames in the P. aeruginosa genome. The surprising primary hit of this analysis was the promoter of an uncharacterized open reading frame, P A 415 7. P A 415 7 is located upstream ofthefep operon, which is known to be involved in iron acquisition. PA4157 shares high homology to the IclR family of transcriptional regulators which are known to regulate quorum sensing (QS), an elaborate cell-cell communication signaling system that uses quoromones. We postulated two hypotheses: 1) AmpR regulation of QS genes is mediated by PA4157, and 2) PA4157 may be involved in iron acquisition. To address the role of P A 415 7 we generated an in-frame chromosomal deletion of P A 415 7 in P. aeruginosa PA01 (PA0 PA4157). We compared PA0 PA4157 with its parent strain P A0 1 for its ability to produce quoromones using Chromobacterium violaceum as an indicator strain and LasA proteases using Staphylococcus aureus. We also tested its role in virulence using a Caenorhabditis elegans killing assay. Growth in iron-deficient media was also examined to determine if P A4157 has a potential role in iron uptake regulation. Our preliminary results suggest that P A 415 7 is not involved in quorum sensing regulation but does seem to exert a negative regulatory effect on iron uptake in P. aeruginosa P A0 1.

Role of AmpR and alginate production in Pseudomonas aeruginosa biofilm antibiotic resistance associated with cystic fibrosis

Pseudomonas aeruginosa is an ubiquitous Gram-negative opportunistic pathogen that is commonly found in nosocomial infections, immunocompromised patients and burn victims. In addition, P. aeruginosa colonizes the lungs of cystic fibrosis patients, leading to chronic infection, which inevitably leads to their demise. In this research, I analyzed the factors contributing to P. aeruginosa antibiotic resistance, such as the biofilm mode of growth, alginate production, and 13-lactamase synthesis. Using the biofilm eradication assay (MBEC™ assay), I exposed P. aeruginosa to B-lactams (piperacillin, ceftazidime, and cefotaxime ), aminoglycosides ( amikacin, tobramycin and gentamicin), and a fluoroquinolone ( ciprofloxacin) at various concentrations. I analyzed the effects of biofilm on P. aeruginosa antibiotic resistance, and confirmed that the parent strain PAO 1 biofilms cells were > 100 times more resistant than planktonic (freefloating) cells. The constitutively alginate-producing strain PDO300 exhibited an altered resistance pattern as compared to the parent strain P AO 1. Finally, the role of AmpR, the regulator of ampC-encoded 13-lactamase expression was analyzed by determining the resistance of the strain carrying a mutation in the ampR gene and compared to the parent strain PAOl. It was confirmed that the loss of ampR contributes to increased antibiotic resistance.

Characterization of L-Serine Deaminase and its Potential Role in the Pathogenicity of Pseudomonas aeruginosa

All pathogens require high energetic influxes to counterattack the host immune system and without this energy bacterial infections are easily cleared. This study is an investigation into one highly bioenergetic pathway in Pseudomonas aeruginosa involving the amino acid L-serine and the enzyme L-serine deaminase (L-SD). P. aeruginosa is an opportunistic pathogen causing infections in patients with compromised immune systems as well as patients with cystic fibrosis. Recent evidence has linked L-SD directly to the pathogenicity of several organisms including but not limited to Campylobacter jejuni, Mycobacterium bovis, Streptococcus pyogenes, and Yersinia pestis. We hypothesized that P. aeruginosa L-SD is likely to be critical for its virulence. Genome sequence analysis revealed the presence of two L-SD homo logs encoded by sdaA and sdaB. We analyzed the ability of P. aeruginosa to utilize serine and the role of SdaA and SdaB in serine deamination by comparing mutant strains of sdaA (PAOsdaA) and sdaB (PAOsdaB) with their isogenic parent P. aeruginosa P AO 1. We demonstrated that P. aeruginosa is unable to use serine as a sole carbon source. However, serine utilization is enhanced in the presence of glycine and this glycine-dependent induction of L-SD activity requires the inducer serine. The amino acid leucine was shown to inhibit L-SD activity from both SdaA and SdaB and the net contribution to L-serine deamination by SdaA and SdaB was ascertained at 34% and 66 %, respectively. These results suggest that P. aeruginosa LSD is quite different from the characterized E. coli L-SD that is glycine-independent but leucine-dependent for activation. Growth mutants able to use serine as a sole carbon source were also isolated and in addition, suicide vectors were constructed which allow for selective mutation of the sdaA and sdaB genes on any P. aeruginosa strain of interest. Future studies with a double mutant will reveal the importance of these genes for pathogenicity.

The role of redox signaling in the molecular mechanism of tamoxifen resistance in breast cancer.

The emergence of tamoxifen or aromatase inhibitor resistance is a major problem in the treatment of breast cancer. The molecular signaling mechanism of antiestrogen resistance is not clear. Understanding the mechanisms by which resistance to these agents arise could have major clinical implications for preventing or circumventing it. Therefore, in this dissertation we have investigated the molecular mechanisms underlying antiestrogen resistance by studying the contributions of reactive oxygen species (ROS)-induced redox signaling pathways in antiestrogen resistant breast cancer cells. Our hypothesis is that the conversion of breast tumors to a tamoxifen-resistant phenotype is associated with a progressive shift towards a pro-oxidant environment of cells as a result of oxidative stress. The hypothesis of this dissertation was tested in an in vitro 2-D cell culture model employing state of the art biochemical and molecular techniques, including gene overexpression, immunoprecipitation, Western blotting, confocal imaging, ChIP, Real-Time RT-PCR, and anchorage-independent cell growth assays. We observed that tamoxifen (TAM) acts like both an oxidant and an antioxidant. Exposure of tamoxifen resistant LCC2 cell to TAM or 17 beta-estradiol (E2) induced the formation of reactive oxidant species (ROS). The formation of E2-induced ROS was inhibited by co-treatment with TAM, similar to cells pretreated with antioxidants. In LCC2 cells, treatments with either E2 or TAM were capable of inducing cell proliferation which was then inhibited by biological and chemical antioxidants. Exposure of LCC2 cells to tamoxifen resulted in a decrease in p27 expression. The LCC2 cells exposed to TAM showed an increase in p27 phosphorylation on T157 and T187. Conversely, antioxidant treatment showed an increase in p27 expression and a decrease in p27 phosphorylation on T157 and T187 in TAM exposed cells which were similar to the effects of Fulvestrant. In line with previous studies, we showed an increase in the binding of cyclin E-Cdk2 and in the level of p27 in TAM exposed cells that overexpressed biological antioxidants. Together these findings highly suggest that lowering the oxidant state of antiestrogen resistant LCC2 cells, increases LCC2 susceptibility to tamoxifen via the cyclin dependent kinase inhibitor p27.

The Role of Ras Oncogene in Differentiation of 3T3-L1 Preadipocytes

Ras is a proto-oncogene that codes for a small GTPase and is responsible for linking several extracellular signals to intracellular mechanisms that involve cell growth, differentiation and cell-programmed death in normal and diseased cells. In all these processes, Ras has been extensively investigated. However, the role of Ras GTPases is still poorly understood during the differentiation of 3T3-L1 preadipocytes. In this study I investigated the role of the H-Ras defective mutant, Ras:G12V on the differentiation of 3T3-L1 preadipocytes. Preadipocytes were differentiated in vitro to adipocytes (fat cells) by adding an induction medium containing several factors including glucose and insulin. The formation of fat cells evidenced by the visualization of lipid drops as well as by quantifying the accumulation of Oil red O into lipid drops. To examine the role of Ras:G12V mutant, several selective mutations were introduced in order to determine the signaling transduction pathways (i.e., PI3(K)kinase and MAP(K)Kinase) responsible for the Ras-dependent adipogenesis. Cells expressing Ras:G12V mutant stimulated 3T3-L1 preadipocyte differentiation without he need for induction media, suggesting that Ras activation is an essential factor required for 3T3-L1 preadipocyte differentiation. Introduction of a second mutation on Ras:G12V (i.e., Ras:G12V;E37G), which blocks the activation of the MAPKinase pathway, strongly inhibited the 3T3-L1 preadipocyte differentiation. It is also important to note Ras:G12V:E37G double mutant does not inhibit the activation of the PI3kinase pathway. Other Ras double mutants (Ras:G12V;S35T, and V12G;C40Y) showed a modest inhibition of the 3T3-L1 preadipocyte differentiation. Taken together, these observations indicate that Ras plays a selective role in 3T3-L1 preadipocyte differentiation. Thus, understanding which specific pathway Ras employs during preadipocyte differentiation could clarify some of the uncertainties surrounding fat production.

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.

Cardiac Neural Crest Cells and Their Role in Murine Purkinje Fiber Development

The heart beat is regulated by the cardiac conduction system (CCS), a specialized group of cells that transmit electrical impulses around the heart chambers. During development, ventricular CCS cells originate from embryonic cardiomyocytes and not from the neural crest. Nonetheless, discoveries in chick implied that the cardiac neural crest (CNC) cells contribute to proper development of the ventricular CCS. In this report, the Splotch mouse mutant (Pax3sp), in which the CNC cells do not migrate to the heart, was used to investigate whether these cells also affect proper CCS development in mammals. Homozygote mutants (Pax3Sp!Sp) are lethal on 111 Embryonic Day 13 (E13), and can be phenotyped by spina bifida and exencephaly. Pax3Spi+ mice were crossed to obtain wild type, Pax3 Spi+ and Pax3 Sp!Sp embryos. Comparison of hematoxylin and eosin stained histological sections showed less trabeculation in El2.5 cardiac ventricles of Pax3Sp!Sp. Furthermore, immunofluorescence analysis with the Purkinje fiber marker Cx40 showed a qualitative difference between wild type and mutant hearts. Quantitative analysis indicated that Pax3 Sp!Sp ventricles had fewer Cx40 expressing cells, as well as less Cx40 being expressed per cell when compared to wild type ventricles. Immunofluorescence with the H3 histome mitosis antibody showed fewer proliferating cells in the ventricles of mutant embryos when compared to controls. These results suggest that CNCC affect the morphogenesis of cardiac ventricles and the development of the ventricular CCS by contributing cellular proliferation.

Understanding the role of Dimethylsulfoniopropionate as a potential chemotactic cue for coral-associated bacteria

Most reef-building corals are known to engage in non-pathogenic symbiosis not only with unicellular dinoflagellates from the genus Symbiodinium, but also with other microscopic organisms such as bacteria, fungi, and viruses. The functional details of these highly complex associations remain largely unclear. The impetus of this study is to gain a better understanding of the symbiotic interaction between marine bacteria and their coral host. Studies have shown that certain bacterial orders associate with specific certain coral species, thus making the symbiotic synergy a non-random consortium. Consequently both corals and bacteria may be capable of emitting chemical cues that enable both parties to find one another and thus generate the symbiosis. The production of these cues by the symbionts may be the result of environmental stimuli such as elevated ocean temperatures, increased water acidity, and even predation. One potential chemical cue could be the compound DMSP (Dimethylsulfoniopropionate) and its sulphur derivatives. Reef-building corals are believed to be the major producers of the DMSP during times of stress. Marine bacteria utilize DMSP as a source of sulfur and carbon. As a result corals could potentially attract their bacterial consortium depending on their DMSP production. This would enable them to adapt to fluctuating environmental conditions by changing their bacterial communities to that which may aid in survival. To test the hypothesis that coral-produced DMSP plays a role in attracting symbiotic bacteria, this study utilized the advent of high-throughput sequencing paired with chemotactic assays to determine the response of coral-associated bacterial isolates towards the DMSP compound at differing concentrations. Chemotaxis assays revealed that some isolates responded positively towards the DMSP compound. This finding adds to existing evidence suggesting that coral-associated pathogens utilize chemotaxis as a host colonization and detection mechanism. Thus the symbiotic bacteria that make up the coral microbiome may also employ this process. Furthermore this study demonstrates that bacterial motility may be a strong contributing factor in the response to the chemotactic cue. Swarming motility may be better suited for bacteria that need to respond to a chemical gradient on the surface of the coral. Therefore the isolates that were able to swarm seemed to respond more strongly to the DMSP.

Transcription-Coupled DNA Supercoiling in Escherichia Coli: Mechanisms and Biological Functions

Transcription by RNA polymerase can induce the formation of hypernegatively supercoiled DNA both in vivo and in vitro. This phenomenon has been explained by a “twin-supercoiled-domain” model of transcription where a positively supercoiled domain is generated ahead of the RNA polymerase and a negatively supercoiled domain behind it. In E. coli cells, transcription-induced topological change of chromosomal DNA is expected to actively remodel chromosomal structure and greatly influence DNA transactions such as transcription, DNA replication, and recombination. In this study, an IPTG-inducible, two-plasmid system was established to study transcription-coupled DNA supercoiling (TCDS) in E. coli topA strains. By performing topology assays, biological studies, and RT-PCR experiments, TCDS in E. coli topA strains was found to be dependent on promoter strength. Expression of a membrane-insertion protein was not needed for strong promoters, although co-transcriptional synthesis of a polypeptide may be required. More importantly, it was demonstrated that the expression of a membrane-insertion tet gene was not sufficient for the production of hypernegatively supercoiled DNA. These phenomenon can be explained by the “twin-supercoiled-domain” model of transcription where the friction force applied to E. coli RNA polymerase plays a critical role in the generation of hypernegatively supercoiled DNA. Additionally, in order to explore whether TCDS is able to greatly influence a coupled DNA transaction, such as activating a divergently-coupled promoter, an in vivo system was set up to study TCDS and its effects on the supercoiling-sensitive leu-500 promoter. The leu-500 mutation is a single A-to-G point mutation in the -10 region of the promoter controlling the leu operon, and the AT to GC mutation is expected to increase the energy barrier for the formation of a functional transcription open complex. Using luciferase assays and RT-PCR experiments, it was demonstrated that transient TCDS, “confined” within promoter regions, is responsible for activation of the coupled transcription initiation of the leu-500 promoter. Taken together, these results demonstrate that transcription is a major chromosomal remodeling force in E. coli cells.