Third Annual Sigma Xi Poster Session
Evidence for the existence of a central class Hox gene in sponges
In contrast to some well-known animal models, very little is known of the developmental control of growth in sponges. This lack of information has limited our understanding of important events in the course of animal evolution. Genes present in animals that were among the first to branch off of the metazoan lineage will likely perform more restricted functions than those found in organisms that diverged later in animal evolution. Therefore, our research is centered around understanding the genetic constituency of developmentally important genes in sponges, and the functionality of morphogenetic regulatory programs in these organisms. In particular, we are focusing on the evolutionary and developmental significance of Hox genes in sponges. To this end, we performed a PCR-based survey and report here the isolation of the first Hox gene from a Poriferan genome. Clustal W placed the putative Hox gene isolated from Haliclona loosanoffi (Hox3Hl) with central class (PG-3) Hox genes and phylogenetic analysis consistently and unambiguously placed the Hox3Hl sequence in the PG-3 group. The Hox3Hl sequence has family-specific PG-3 amino acid residues that include F, R, C, P, and M at positions 2, 4, 7, 9 and 14 respectively. This finding is surprising given that only anterior and posterior class Hox genes have been found in cnidarians. Additionally, we used RT-PCR analysis to examine the expression profile of this Hox gene in developing sponge tissues. We show that the gene is expressed in free-swimming larvae as well as attaching and settling larvae. This data may provide significant insights into early animal evolution.
Sara Brady and Allison Schaffer
Preliminary investigations of shelter competition among the Asian shore crab and native mud crabs
This study examined the potential impact of the recently introduced Asian shore crab, Hemigrapsus sanguineus on shelter utilization by two native species of mud crabs, Eurypanopeus depressus and Panopeus herbstii, using laboratory experiments and field sampling at two sites in western Long Island Sound (Black Rock Harbor, BRH; Milford Harbor, MH). Abundance and distribution patterns of these species differed at the two sites. Similar numbers of mud and Asian crabs were found under rocks at BRH, but Asian crabs outnumbered mud crabs 15:1 at MH. Asian crabs were most abundant at mid-tide level, whereas 90% of the mud crabs occurred low in the intertidal. This is likely due to the low tolerance for dessication exhibited by xanthid crabs (Grant and McDonald, 1979). At low tidal elevation, where most of the overlap occurred, between-site differences in under-rock microhabitat utilization were present. Only mud crabs were found beneath 75% of the rocks sampled at BRH, but at MH, mud crab species alone were found under only 5% of the rocks. Relative crab densities likely affect competitive outcomes and ultimately space utilization patterns. Results of shelter competition experiments conducted in the laboratory did not support the hypothesis that H. sanguineus affects shelter utilization by native mud crabs. The percentage of mud crabs occupying shell shelters remained unchanged when Asian crabs were present, but the percentage of Asian crabs occupying shell shelters decreased relative to controls in trials where mud crabs were present. These findings suggest that E. depressus and P. herbstii may affect patterns of habitat use by H. sanguineus, especially in the lower intertidal, where these species occur together. However, direct experimental manipulations in the field coupled with long-term monitoring are needed to fully understand the role of competitive interactions in determining the local distribution of these species.
Poster Presentation: 23rd Annual
Induction of metallothionein in articular chondrocytes grown with steroid or TGFB supplementation
Human articular chondrocytes were grown in Dulbeccos Modified Eagle Medium (DMEM), DMEM supplemented with the steroid dexamethosome or DMEM containing differentiation factor TGFB. The three sets of chondrocyte were harvested and studied for the short-term effects of the toxic metal ion Cadmium on cellular protein, DNA and the metal binding protein metallothionein (MT). DNA levels were increased in cells grown with TGFB and decreased when grown with dexamethosome. TGFB dramatically increased cellular protein levels relative to control and dexamethasome treated cells. Short term (48 hr) treatment of cell culture with cadmium resulted in a dose-dependent induction of MT. MT induction was highest in cells grown with dexamethasome and lowest in cells grown with TGFB. The results suggest that MT expression is effected by differentiation of the chondrocytes caused by the media supplements tested.
Kimberly A. Cullen
Co-researchers: R.M. Damian Holsinger, Janetta G. Culvenor, Karolina Röyttä, Colin L. Masters and Geneviève Evin, Department of Pathology, The University of Melbourne and the Mental Health Research Institute, Parkville, Victoria 3010, Australia.
Redefinition of BACE transmembrane domain to include residues 455-460
Alzheimer's disease β-secretase, BACE, is a novel aspartyl protease with a single transmembrane domain located in its C-terminal region. To produce soluble BACE, we have expressed a construct corresponding to BACE putative ectodomain (residues 1 -460) in CHO-S mammalian cells. Unexpectedly, the protein was not secreted but was recovered in cell lysates, as determined by both Western blotting and assay for β-secretase activity. Biochemical, and microscopic studies of the cells indicate that BACEPD is associated with membranes. Our data suggest that BACE transmembrane domain extends to residues 455-460, a finding supported by Kyte-Doolittle hydrophobicity analysis of BACE transmembrane region.
Abbreviations: AD: Alzheimer's disease; APP: amyloid precursor protein; BACE: β-site APP-cleaving enzyme; BACEPD: BACE protease domain; CHO-S: Chinese hamster ovary suspension cells; RT-PCR: reverse transcription-polymerase chain reaction; TMD: transmembrane domain.
Christine Curley and Christine McDonagh
Molecular strategies for identifying species boundaries in marine invertebrates: a sequence-based approach
Marine invertebrates that are broadcast spawners (i.e.,
release gametes into the water) would seem to have genetically well-mixed
populations with broadly dispersed larvae. This would lead to genetic
homogeneity among populations, thus, reducing the opportunity for speciation to
occur via traditional mechanisms (e.g.,
physical isolation). Despite these apparent barriers to speciation events, we
have good evidence of speciation rates similar to, or higher than, those on
land, which raises important questions about how speciation occurs in marine
systems. We are interested in determining whether a polytypic sponge from the
Sean Harrell and Eric Portante
Research Advisor: Michael Brienza
Using physiology to teach physics
Our research consists of finding ways to use human
physiology to teach physics. Since June 2002, we have been working on several
specific experiments: how phase shifts are used in human auditory localization,
and how the eye images objects. These experiments are just moving into one of
the final testing phases: implementing them in one section of the General
Physics Laboratory. Our research will continue this summer and most likely into
next semester: we will be attending a conference at
Effects of light availability on chemical defenses of three deciduous tree species
Plants have evolved a sophisticated array of chemical defenses as protection from attack by insect herbivores. Availability of resources can affect how plants allocate limited resources to defenses, versus to growth or maintenance needs. To address this idea, we used a combination of field and lab experiments to determine variability in defensive chemistry among plants and subsequent effects on the herbivores that eat those plants. We investigated the effects of light availability on resource allocation to chemical defenses for saplings of three deciduous temperate forest trees (Red Maple, Black Cherry and Sassafras) in mid-summer. We bioassayed the foliage by feeding the larvae of the promethea moth on leaves from those trees. Contrary to our predictions, the caterpillars grew better and faster on foliage from trees growing under high light. Few studies have investigated plant defenses in mid-summer, as we did. Much of the theoretical and empirical work conducted previously focuses on plant allocation to defenses in the spring, when plant growth and insect abundance is highest. It is our feeling that food quality (availability of protein, water, etc…) may have played an important role in determining herbivore performance and overwhelmed the signal of defenses in the plants. Pending chemical analyses of the foliage will offer insight into the relative levels of defenses in these plants and will help us determine how our results fit with the predictions of current plant defense theories.
Factors contributing to the presence of algal blooms in
Estuaries are the highly biologically, chemically, and
physically active systems where freshwater and seawater mix. In these systems, phytoplankton,
microscopic organisms capable of primary production, serve as the base
of the food chain. However, in some
cases, phytoplankton can grow to levels that are detrimental to the system in
which they are located. These algal
blooms impair recreation and can cause more serious problems as they
decompose. Bacteria that decompose the
algae use oxygen and oxygen levels following algal blooms drop to levels that
can kill fish and shellfish. Our
objective was to identify factors contributing to the presence of algal blooms
in the lower
Nora Lopez and Kimberly Young
Chemical and structural defenses in four species of temperate sponge against the hermit crab Pagurus longicarpus
The majority of studies examining anti-predator defenses in sponges have focused on tropical species, and these studies have found chemical defenses to be extremely effective at deterring predators. Physical defenses have been found to be relatively ineffective, but few studies have looked for interactions between physical and chemical defenses in sponges. We examined anti-predator defenses in four species of temperate sponge from Long Island Sound using a common hermit crab (Pagurus longicarpus) as a predator. We identified potential synergistic interactions between structural and chemical defenses in Microciona prolifera. Structural defenses were found to be effective in Cliona celata whereas artificial food containing chemical extracts from Haliclona loosanoffi, but showed no preference for spicule-laden artificial food. Chemical extracts from Halichondria bowerbanki deterred predation, however in contrast to many tropical sponges, spicules were also deterrent. No evidence of a synergism was observed in the H. bowerbanki feeding trials. These preliminary results indicate that temperate sponges may use both chemical and structural defenses against a potential crab predator. In addition, physical and chemical defenses in M. prolifera may interact synergistically. We are currently examining additional aspects of both chemical and structural defenses for adult and larvae phases of the sponge life cycle including an identification of specific predators and an examination of levels of predation under field conditions.
River influences on estuarine Enteromorpha intestinalis
Freshwater inputs from
rivers and streams alter salinity of coastal estuaries, and are also important
conduits for the delivery of nutrients such as nitrogen and phosphorus. Changes in these environmental factors may
impact the growth of organisms in estuaries.
We studied the impact of freshwater inputs on primary producers in the
Serial APACHE II scores in patients with prolonged intensive care unit stay
The purpose of this study was to assess the performance of serial acute physiology and chronic health evaluation (APACHE II) scores in predicting outcome of Intensive Care Unit (ICU)patients (PTS) with an ICU length of stay (LOS)>30 days. A prospective observational study over a period of fifteen months was conducted in a 40 bed medical-surgical ICU at a tertiary care teaching hospital. Demographic data were collected using Project IMPACT (PI). APACHE II scores were recorded on admission and weekly for four weeks in PTS with a LOS>30 days. Survivors were those PTS who were alive >two months after discharge from the hospital. The data were analyzed using Student's t test. Data from 2,878 PTS were evaluated. Thirty-seven PTS had an ICU LOS >30 days (1.2%) and utilized 15% of the total ICU bed-days. Fourteen PTS died in the hospital or within two months of their discharge. Of the remaining 23 PTS, 19 were discharged to skilled nursing facility (SNF) and 4 PTS returned home. There was a significant difference in the age of the survivors versus (vs) the non-survivors (53.5 vs 72.8, p=.001). The mean admission APACHE II scores between the two groups were equal (22.5 vs 22.6). After one week, the mean APACHE II scores of the survivors vs the non-survivors differed (15.2 vs 18.5) and reached statistical significance on weeks 2(15.8 vs 19.3, p=0.03), 3 (14.5 vs 20.1, p=0.004) and 4(12.9 vs 21.1, p=0.0006). One year follow up revealed 3 PTS at SNF, 17 PTS at home, 1 PT expired, 2 PTS lost to follow-up. Serial APACHE II scores may be useful in predicting outcome of patients with prolonged ICU stays. Further evaluation of serial APACHE II scoring appears warranted.
Expression of the Pax 3/7-like gene in Marine Sponges
Pax genes belong to a family of transcription factors that encode multiple and diverse DNA-binding domains. Pax family members are involved in the development and control of the central nervous system and other organs including the kidney, pancreas, and eye in triploblastic organisms. A few Pax family members have been identified in diploblasts (cnidaria and sponges) but their ancestral functions are not yet resolved. We report here the expression analysis of a Pax 3/7 family member in marine sponge tissue. We also present phylogenetic data that may aid in the understanding of the evolutionary diversification of the Pax gene family. The presence of Pax gene sequences in sponges (which lack nerve cells or any type of nervous system) presents an opportunity to determine their function before tissues evolved in animals.
The effect of T3, TGF-Beta, and Dexamethasone on the growth and development of the normal human articular chondrocyte extracted from the knee
The purpose of this experiment was to determine the effect that different growth factors and hormones had on human chondrocyte development and growth. The human condrocytes used were derived from normal human articular cartilage found in the knee (NHAC-kn) supplied from the Clonetics Chondrocyte Cell System, a system that quickly generates the NHAC-kn for experimental application in research. The growth factor TGF-Beta (TGFB) and the hormones dexamethasone (Dex) and triiodothyronine (T3) were used in this experiment. Their effect on the chondrocytes' ability to produce protein, on the cells' levels of DNA, and on the alkaline phosphate activity of the cells, were recorded. Assays were used to determine levels of protein, DNA, and alkaline phosphatase in the chondrocytes. The Lowry assay was used to determine the ug of protein the chondrocytes produced per dish. The DNA assay was used to determine the concentration of DNA in ng the chondrocytes produced per dish. The alkaline phosphate with the Lowry assay was used to determine the amount of enzyme produced per mg of protein was produced in each dish by chondrocytes living in different growth factors and hormones. For each treatment and for different days, the results were graphed from these three assays so that comparisons could be made and the effect of the growth factors and hormones could be determined.
After this experiment was performed and the results recorded and observed, a second experiment was performed involving different concentrations of TGFB. The cells were grown in different concentrations of TGFB to determine at which concentration the most protein, alkaline phosphates, and DNA was produced. Chondroytes are the only cell component of cartilage. Hyaline or articular cartilage provides protection in synovial joints, the most abundant type of joint in the body, by covering opposing bone surfaces and absorbing compression and stress placed on the joint through use. The cartilage prevents the bone ends involved in the joint from degrading or eroding due to friction when the bone ends rub together. It is tough, flexible, and avascular (containing no blood vessels). Condrocytes are essential for the preservation of cartilage because they produce and maintain the extracellular matrix of cartilage, which has the capacity to bear the weight planted on the joint. Articular cartilage is greatly researched because of its direct involvement in arthritis and joint injuries, which can cause disabilities.
Katharine E. Silva
Dr. Cathleen Wigand, Environmental Protection Agency, Atlantic Ecology
Effects of nutrient enrichment of Spartina patens on Chlorophyll Levels
This experiment examined the chlorophyll levels in leaves of the salt marsh grass, S. patens. Chlorophyll levels can be correlated with amounts of photosynthesis taking place within a plant. Samples were taken from each plot. Pigments extracted were run through an HPLC instrument for calculation of chlorophyll a and b levels. The hypothesis was that S. patens under +N-P and +N+P treatments (specifically +N-P due to the high level of nitrogen without the abundance of phosphorous) would have the highest increase in rates of photosynthesis. Predictions were that the chlorophyll a and b levels would be higher in the samples enriched with +N-P and +N+P treatments. Treatments +N+P and +N-P showed to have the highest levels of both chlorophyll a and b. Treatment +N+P had the highest of all the treatments. Treatment -N+P had the lowest levels of both chlorophyll a and b. The control (-N-P) levels were in between the highest and lowest levels. The experiment's results provide data to support the hypothesis. The nitrogen enriched patens showed higher levels of chlorophyll. Chlorophyll levels correlate with the photosynthesis rate of plants; the higher the chlorophyll level, the increase in photosynthesis, thus an increase in glucose produced.
Nicole E. Sparling and Marina C. Simeone ’02
Characterization of the antioxidant protein 2 (Aop2) gene family: Differential regulation in the oxidative stress response
Aop2 is a unique member of the thiol-specific antioxidant family of proteins known to reduce reactive oxygen species in the presence of thiol-containing electron donors. It is also a candidate atherosclerosis gene in mice. An analysis of Aop2 expression has revealed the existence of multiple transcripts not previously reported, each with a unique tissue distribution. We have also determined one of these to be the highly related intronless gene Aop2-rs1, which appears to be exclusively expressed in testes. Additionally, an Intron 1 specific probe identified two alternative transcripts not identified with the Aop2 specific probe. Treatment of a murine hepatocyte cell line with glucose oxidase led to the specific and transient induction of the primary 1.47 kb transcript, while serum deprivation and restimulation regulated the expression of another transcript in a time dependent manner. The effects of high fat diet on expression of these transcripts in heart and liver in atherosclerosis susceptible mice have also been analyzed. Lastly, expression and purification of each encoded protein is allowing for functional characterization of their antioxidant activities. Based on distinct transcriptional regulation, our data suggest that these alternative transcripts may play varying roles in response to oxidative stress and signaling pathways.
Rate of enantiomeric conversion of helical peptides measured by dynamic NMR spectroscopy
Homo-oligomers of the amino acid alpha- aminoisobutyric acid (AIB) are known to form 310-helicies in solution. These helical peptides, stabilized by intramolecular hydrogen bonds, exist in a dynamic equilibrium between two equally populated conformations:left-handed and right-handed helicies. We have employed dynamic NMR spectroscopy to measure the rate of interconversion between the enantiomeric helicies in peptides six and eight residues in length. Our hypothesis states that the octamer will have a slower rate of interconversion than the hexamer due to two more hydrogen bonds that stabilize its helix.
Expression analysis of a BarH1 class gene in developing Halichondria larvae
We previously reported the isolation of a novel, Antennapedia-class, BarH1-like gene from screens of a marine sponge Halichondria cDNA library. Here, we investigate the expression of that gene from both developmental time specific and spatial perspectives. Specifically, we employed RT-PCR techniques to look at expression across six different larval developmental stages and adult disaggregated and re-aggregating tissue. We found that the gene is highly expressed in newly released larvae (presumably a maternal message), after which expression is down-regulated. The gene is again expressed in late stage free-swimming larvae up until they have settled and attached, where expression is again down-regulated. Low levels of expression are observed in adult tissue with little difference in expression between disaggregated and re-aggregated tissues. We also performed in situ hybridization to examine where the gene is expressed in adult tissue (housing developing embryos) and developing larvae. Embryos and free swimming larvae exhibit high levels of BarH1 expression while adult maternal sponge tissues show little expression. In situs to free swimming larvae show BarH1 expression to be concentrated toward the posterior end, with a gradient toward the anterior. This polarity of expression during early development has been observed with antennapedia-class homeobox genes in other animals. Our discovery of both the existence of this type of gene in sponges and its interesting developmental expression pattern may give us insight into the evolutionary conservation of animal body plans.
Jennifer Torp, Jeffrey Tetrault, Derek Bickhart
Faculty Mentors: Olivia Harriott, Malcolm Hill, and April Hill
Molecular analysis of the bacterial community in the marine sponges Chondrilla nucula and Microciona prolifera
Studies have shown that marine
sponges harbor a tremendous diversity of microorganisms. The benefits provided by bacterial symbionts are thought to include nutrient assimilation,
structural support and protection of the sponge from disease causing
microbes. It is difficult and sometimes
impossible to grow and study bacterial symbionts in a
lab due to their dependency on the host.
Our work focuses on the molecular characterization of bacteria
associated with the marine sponges Chondrilla nucula and Microciona prolifera. In
this study, total sponge community DNA from C.
nucula and homogenized M. prolifera tissue was used as the source of symbiont DNAs. Bacterial 16S rRNA sequences were amplified by using primers selective
In both the C. nucula
and M. prolifera
amplifications, a single PCR product of approximately 1500 base pairs was
obtained and cloned. Eleven of the
twenty-nine M. prolifera
clones contained the appropriately sized insert, as well as forty-two of the
forty-three C. nucula
clones. Amplified ribosomal DNA
restriction analysis (ARDRA) of desirable clones was done with HhaI and Hpa II, and the banding patterns suggested
that at least 15 pattern were present. The four unique M. prolifera and eleven C. nucula clones
were partially sequenced and at least 500 base pairs of each clone were
analyzed using BLAST, a web-based DNA sequence analysis program of the
Caitlin A. Vida and Kara A. Showalter,
Advisor: CVS Robert S. Seamans Class 183 and Chief Scientist-Gary Jaroslow
in sediment patterns related to El Niño and climate
change in the southern
The purpose of this
study was to examine climatic variations reflected in varve
patterns from sediment core samples taken within the anoxic region of