John R. Porter, Ph.D.

(I am functioning as an Administrative Fellow during the 2005-2006 academic year, so I am not teaching this year.  However, my research continues, and I have a very active laboratory.)

My usual course load includes:

• Evolutionary Biology (BS 270)
• Cell Biology (BS 461)
• Biological Sciences Seminar (BS 493-4)
• Advanced Pharmacognosy (BS 736)*
• Cell Biology Methods and Laboratory (BS 763, BS 767)
• Advanced Ethnobotany (BS 840)*
• Current Research in Pharmacognosy (CH 870)*
• Graduate Seminar (BS 898)

* co-taught with Ara DerMarderosian

The research in my laboratory focuses on three main themes: Natural Product Biosynthesis, Novel Bioassays for Natural Product Development, Natural Products Chemistry, and Agrobacterium-Plant Interactions. These four areas are linked through the emphasis on natural product analysis.  The work described below involves many researchers, including a technician and students at the undergraduate (BS) and graduate (MS, PhD) levels.  Recent students who have graduated from these studies include Nisit Kittipongpatana (natural product biosynthesis, PhD), Joanna Pols (natural products chemistry, PhD), Amy Eyberger (endophyte fungi for biosynthesis, MS), Rajeswari Dondapati (molecular identification of endophyte fungi, MS), Joseph Newcome (molecular identification of endophyte fungi, MS), and Sharon Moravec (antimicrobial activities of natural products, BS).  Current students include Frank Zydel (natural products biosynthesis, PhD student), Lawrence Han (biosynthetic pathway transfer, BS candidate), Melissa Tursiella (biosynthetic pathway transfer, BS candidate), Nirmal Trivedi (biosynthetic pathway genetic analysis, BS candidate), and Laura Flint (tuberculosis assay development, BS candidate).  My technician is Muta Mathai (MS) working on optimization of growth and production by endophyte fungi.

Many compounds have interesting or valuable activities but are in short supply because the organisms producing them are too rare, grow too slowly, or are geographically or politically inaccessible. We have been experimenting with production of compounds through culture of the fungi and bacteria being examined for antimicrobial and anticancer activities. Our main focus is the production of podophyllotoxin, an extremely valuable natural product, through fungal culture.  Through the MS studies of Amy Eyberger, we have discovered two plant endophyte fungi that produce detectable amounts of podophyllotoxin, and we are pursuing this through culture optimization.  These fungi, however, grow very slowly and the product yields have been low to date.  Besides rather traditional means of fungal culture, we are also looking at the possibility of moving one or more parts of the biosynthetic pathway to more tractable organisms.  This gives the work a much more "molecular" flavor.

For a number of years, we have been exploring the possibilities of producing natural product compounds through culture of plant roots initiated with Agrobacterium rhizogenes. A. rhizogenes induces the growth of roots at infection sites through transfer to genetic material (the T-DNA) from the bacterium to the host plant. Genes activated after the transfer alter the plant hormone environment to induce the initiation of roots. This is similar to the callus production following A. tumefaciens transformation, but the A. rhizogenes system is much less studied. The induced and transformed roots can be separated from the source plant and inoculated into defined medium, where they grow independent of plant hormones. Manipulation of the culture environment allows us to study the production of plant natural product compounds in the defined culture. A recent doctoral graduate, Dr. Nisit Kittipongpatana, studied two such systems, the production of solasodine by root cultures of Solanum aviculare and the production of valepotriates by root cultures of Valerianella locusta. Solasodine can be used for the production of steroid hormones. The valepotriates are thought to be the relaxation- and sleep-inducing compounds of species such as valerian. For both species, we have found that the carbon source, medium composition, and the presence of stress-inducing elicitors have an influence on natural product production. These have served as interesting model systems to further our understanding of optimization of natural product production and root induction in various species.

Novel Bioassays for Natural Product Development

While there are a number of assays available, especially for discovery of antineoplastic and antimicrobial compounds, many disease states lack a suitable assay to help speed drug development.  Assays, whether biological (whole-cell) or biochemical (purified enzyme or receptor) in nature, are continuously in development in many laboratories.  We have begun focusing on the development of a novel biochemical assay for discovery of anti-tuberculosis drugs.  Current tuberculosis assays are difficult because of the slow growth of the organism, hazards of working with the infectious agent, and the unreliability of the several model organisms in providing clinically-applicable results.  We are taking advantage of the unique protein processing of the tuberculosis organism to develop assays that will lead to new drugs.  Cloning of several tuberculosis bacterium genes into alternate hosts will reduce the danger to the researchers and lead to an assay with a known mechanism and high specificity to the target pathogen.  This takes advantage of the knowledge gained through complete sequencing of the Mycobacterium tuberculosis genome, although we will also examine sequences and gene structures in two model mycobacteria, M. smegmatis and M. terrae.  We expect this assay to allow us to screen a large battery of compounds and extracts for specific anti-tubercular activity as leads to further drug development.  It is possible that compounds discovered will also show activity against the pathogen for leprosy, M. leprae.

The vast diversity of biological organisms has led to a comparably vast array of chemically complex natural products. Since only a small portion of the world’s organisms have been examined chemically, we are working under a fundamental hypothesis that there are still many molecules to be discovered that will be found to have interesting biological activities. Although the number of possible molecular targets is enormous, we focus in part of our work on molecules which have antimicrobial and anticancer activities. Through our collection and culture activities, we have developed a biological library of organisms from the plant, fungal and bacterial kingdoms. We are in the process of determining the potential of extracts from these organisms to inhibit growth of a collection of clinically-relevant organisms which have been particularly problematic in the immunocompromised patient population, including those undergoing chemo- or radio-therapy for cancer, those who are immuno-suppressed due to organ transplant, and those with acquired and hereditary immune system pathologies. We also examine the extracts for potential anticancer activity through cytotoxicity and cell-growth assays.

An even larger collection of extracts is available through our collaboration with the National Cancer Institute Developmental Therapeutics Program collection of organism extracts. Covering mostly plant, microbial and marine organism extracts, we have focused most of our work so far on the organic plant extracts. Dereplication of the diversity of plant species allows us to focus on those species which have received almost no chemical work previously. Recent work by a doctoral student, Dr. Joanna Pols, led to discovery of several compounds with antimicrobial activity.

                                                    g.

The diterpenes 19-trachylobanoic acid (a), 8, (14)15-pimaradien-18-oic acid (b), 16-kauren-19-oic acid (c), and 7,15-pimaradien-18-oic acid (d), the polyacetylenes 13(E)-octadecadiene-9,11-diynoic acid (mitrephoric acid) (e) and 17-octadecene-9,11,13-triynoic acid (oropheic acid) (f) isolated from Mitrephora celebica (Annonaceae), 6-stearolic acid (g) from Pentagonia sp. nov. (Rubiaceae), and the resveratrol oligomers hopeaphenol A (h), isohopeaphenol A (i), and vaticaphenol (j) isolated from Vatica oblongifolia ssp. oblongifolia (Dipterocarpaceae). Compounds a, b, e, f, g, h, and j have antimicrobial activity. Compounds c, d, h, and i were not previously known.

Agrobacterium-Plant Interactions

A. rhizogenes  (syn. Rhizobium rhizogenes)is important to our work with root cultures for plant natural product production. Studies begun several years ago led to the preparation of a review on A. rhizogenes host range and virulence. Our work showed that the host range of A. rhizogenes was much broader than had been previously thought. We have also conducted studies that have implicated the hormonal and molecular environment of the plant-bacterium interaction, rather than events such as growth of the bacterium, chemotaxis, and plant cell wall binding, as limiting factors in the development of a successful infection.

Although there is considerable literature on A. tumefaciens (syn. R. radiobacter) and it is generally presumed that the A. rhizogenes system is similar, relatively little is known about the specifics of the plant interaction and molecular biology of the root-inducing species. The A. tumefaciens genome has been almost completely sequenced, but only a small fraction of the A. rhizogenes genome is known (primarily genes on the pRi plasmid). A comparison of the genes of both A. rhizogenes and R. leguminosarum to the chromosomal virulence (chv) genes of A. tumefaciens, has been conducted using PCR to isolate and amplify the genes with sequence similarity to the A. tumefaciens gene set.  DNA sequence analysis is giving us clues about the similarity and potential functions of these genes in the three organisms. These studies, similar to those in other laboratories, are demonstrating that A. tumefaciens and A. rhizogenes are not nearly as related as once thought.  In fact, several of the genes examined show closer affinity between A. rhizogenes  and R. leguminosarum.  This work will serve as the launch point for the development of additional primers for other A. rhizogenes genes and to help us in our efforts to map the gene locations using pulse-field gel electrophoresis and restriction endonuclease digestion. Our initial hypothesis was that the genome organization of A. rhizogenes is similar to that of A. tumefaciens.  We now have the data that makes us doubt that hypothesis.  Further studies will give us a clearer idea of the affinities within this taxonomically-complex group and lead to a better understanding of A. rhizogenes, an organism useful in natural products synthesis work.

We gratefully acknowledge the Department of Biological Sciences, the Department of Chemistry & Biochemistry (University of the Sciences in Philadelphia), the Pardee Foundation for Cancer Research, and GlaxoSmithKline  for significant research funding.

MAJOR SERVICE ACTIVITIES

• Administrative Fellow
• Biology Liaison, Science and Technology Center
• Medals Judge and Advisory Board Member, Delaware Valley Science Fairs
• Webmaster, American Society of Pharmacognosy