Expression of Neuronal Isoforms of the Cytoplasmic Dynein and Dynactin in B35 Neuroblastoma Cells.
Jackelyn
K. Burke and Kevin T. Vaughn
Cytoplasmic dynein and dynactin are essential in most cells but are highly specialized
in neurons where they are thought to play important roles in development and
function. Work on these proteins has been performed in cultured fibroblasts,
but would benefit from a more neuronal culture system that retains the advantages
of gene manipulation, transfection, imaging, and live-cell assays. We
have established the B35 neuroblastoma cell line which can be propagated in
a non-differentiated state and transfected but can also be induced to differentiate
through dibutyryl-cAMP treatment. Within five hours of differentiation,
these cells adopted a more neuronal morphology with long thin processes resembling
axons and growth-cone-like branching at the ends of these projections. Protein
extracts from parental and differentiated cells were probed by western blot
analysis with antibodies against the cytoplasmic dynein intermediate chains
(ICs) and the p150 Glued subunit of dynactin. Both ubiquitous and neuron-specific
isoforms of the ICs were expressed, as well as ubiquitous and neuronal splicing
variants of p150 Glued . The neuronal forms of both proteins were enhanced
in the differentiated cultures. Immunofluorescence staining with the same antibodies
revealed prominent expression of both complexes in the cell bodies and processes.
Synaptic proteins syntaxin and synaptophysin were up-regulated in differentiated
cells, and immunofluorescence microscopy suggested vesicular staining patterns.
In the presumptive growth cones, tip-tracking of GFP- p150 Glued and
GFP-EB1 was observed, revealing the presence of dynamic microtubule plus-ends
and regulatory systems. Based on novel isoform complexity, we designed new primers
for RT-PCR analysis of IC and p150 Glued transcripts to assess changes
associated with differentiation. These results suggest that B35 cells express
neuron-specific isoforms of cytoplasmic dynein and dynactin and can serve as
a model system for functional analysis of these neuronal proteins. (Supported
by the NSF-REU program)
A Comparison of SNP, SSCP, and Microsatellite Markers in Aedes aegypti using a field population from Trinidad
Julia Adams and Dr. David Severson
The mosquito Aedes aegypti , found in tropical and subtropical regions, is the number one vector for dengue viruses. Because tens of millions of people are infected with the viruses each year, it is important to gain an understanding of dengue vector competence in Aedes aegypti . We looked at three different marker types in regions of the Ae. aegypti genome believed to be associated with dengue vector competence in a field population from Trinidad. We hoped to compare the genotypes and allele frequencies of the mosquitoes collected from dengue positive and negative house sites, to determine which marker type provided the finest resolution of families, and to study how results from each of the different marker types in the Ae. aegypti genome could be utilized. In an attempt to answer these questions, we genotyped the population at six single nucleotide polymorphism (SNP) markers, six single stranded conformational polymorphism (SSCP) markers, and three microsatellite markers. From the data collected, we were able to determine if the markers were in Hardy-Weinberg equilibrium and the percent of each marker type that was polymorphic. Our results are a preliminary screen of the utility of SNP, SSCP, and microsatellite markers in our field population.
Expression of Indoleamine 2, 3-dioxygenase (IDO) during Murine Plasmodium Infection
Protozoan parasites of the genus Plasmodium have long been a health issue of humans. Ancient writings describe an ailment with a seasonal periodicity and deadly outcomes that was most certainly malaria. Today, malaria is primarily a disease of individuals who live in poor countries, and have little access to adequate healthcare. For centuries, a simple extract from Cinchona trees served as an ample treatment, and later insecticides helped control mosquito populations. However with the quick emergence of drug and pesticide resistance in the 20 th century, there is now a strong emphasis on the development of an effective malaria vaccine to control Plasmodium .
Natural malaria infections arise through the introduction of Plasmodium sporozoites into the hosts via mosquito saliva. The liver stage of malaria is the primary stage of infection, and eventually leads to the production of merozoites, which initiate the blood stage infection. The host responds to the blood stage infection by inducing a T cell response. T cells are activated in order to help the hosts immune system battle a variety of pathogens, including viruses, fungi, bacteria and protozoa viral and fungal infections. Malaria is among a large group of chronic infections, including Leishmaniasis and tuberculosis. One of the prominent reasons for the chronicity of these infections is the diminished ability of T cells to proliferate and effectively eliminate the pathogen. Indoleamine 2, 3-dioxygenase (IDO), the rate limiting enzyme in the degradation of tryptophan, reduces T cell effectiveness.
There are two biological roles of IDO: the first is to suppress microbial infections by reducing the availability of the amino acid, tryptophan, in infected tissues. IDO inhibits the replication of intracellular pathogens by depleting tryptophan levels. The second function of IDO deals with the expression of interferon-gamma (IFN). As T cells are activated by macrophage and dendritic cells, INF is expressed by the T cells, which leads to the activation of more macrophage and dendritic cells. When the levels of INF are exceedingly high, the expression of IDO is induced in order to prevent immuno-pathology, due to an over activation of T cells. In response to the reduction of tryptophan, T cell proliferation is arrested between the Go-S phases of the cell cycle, which can lead to T cell death by apoptosis. We hypothesize that due to the increase of IDO expression, a host could be more susceptible to infection because of the inhibition of the T cell response in the liver.
Characterization of 2La Inversion Breakpoints in the Anopheles gambiae Complex
The 2La inversion is hypothesized to increase vector competence endowing species within the Anopheles gambiae complex the ability to live in atypical arid environments. The molecular characterization of the 2La inversion within the Anopheles gambiae complex may reconstruct the evolution of the 2La inversion within the An. gambiae complex, and may in the future contribute in an effort to control the vector in its native habitat. The distal breakpoint of the 2La inversion in An. gambiae Sua has previously been cloned and sequenced. In situ hybridization revealed a BAC clone that crosses the 2La inversion breakpoints. A probe was made from this BAC clone and used to screen the lambda Dash II An. gambiae s.s. (Sua) and the lambda Dash II An. merus phage genomic libraries, two different species in the An . gambiae complex, for clones across the 2La inversion breakpoint. The proximal 2La inversion breakpoint in An. gambiae (Sua) has been recovered, and the recovery of 2La inversion breakpoint clones in An. merus is in progress. Primers were designed using the known Sua breakpoint sequence and used to PCR screen the An. gambiae Bamako phosmid library for inserts along the 2La inversion breakpoints. BLAST search of end sequences of six positives from the PCR screen revealed phosmid inserts along both breakpoints of the Bamako library. Both the proximal and distal 2La inversion breakpoints in An. gambiae were recovered. These 40kB inserts from both distal and proximal 2La inversion breakpoints in the phosmids are currently being sequenced using the new breakthrough Genome Priming System that utilizes transposable elements. A transposable element of known sequence is randomly inserted into the phosmid serving as a basis to attach primers for sequencing. 200 different insertions are selected and sequenced. This process on average generates a 700 bp sequence of the phosmid from each side of the transposable element resulting in a net 1400bp sequenced fragment which facilitates the assembly of the 40Kb insert contig.
Effects of Soy Isoflavones on Cellular Lipid Metabolism
Timbrala
Marshall, Eimear Mullen, and Dr. Neil Shay
The consumption of soy is known to reduce blood cholesterol levels. Isoflavones
are phytochemicals naturally present in soy, and may be responsible, at least
in part for these positive changes. Sterol Regulatory Element Binding
Proteins (SREBPs) are a family of membrane-bound transcription factors that
regulate genes involved in cholesterol biosynthesis and LDL uptake. We
have previously shown that their activity is increased in the presence of soy
isoflavones.
My study is a continuation of previous work. I used transient transfection
assays to examine the effects of increasing doses of isoflavones on SRE directed
gene expression, specifically, expression of the HMG CoA synthase promoter.
The isoflavones I used for these assays were genistein and daidzein. Western
Blots were performed to look at LDL receptor protein levels in response to isoflavone
treatments. Assays were performed to examine cholesterol levels in cells.
The transfection assays showed that the expression of the HMG CoA synthase promoter
increased as isoflavone concentrations increased to a peak of 0.5ul/ml. The
results from the cholesterol assays showed that cells treated with soy extract
or isoflavones were similar to cells treated with cholesterol. This experiment
was only performed once so further analysis is needed.
Determining the Functional Importance of Two MAEBL Isoforms for Malaria Sporozoite Invasion of Mosquito Salivary Glands
Matthew C. Medeiros and John Adams
Malaria ( Plasmodium sp .) is a protozoan blood parasite with a complicated life cycle that progresses through vertebrate and mosquito hosts. The invasion of mosquito salivary glands by Plasmodium sporozoites, an essential step for continued transmission to susceptible vertebrate host, is dependent on the expression of MAEBL. Previous studies within the lab indicate that maebl mRNA undergoes alternative splicing to produce two main open reading frames. ORF1 encodes for a consensus type one membrane protein. ORF2 is created by an alternative splicing junction in intron 3 located 17 nucleotides before the splicing junction observed in ORF1. The alternative splicing junction generates a frame shift in the mRNA introducing a premature stop codon. The stop in translation creates a soluble protein with a unique C-terminus without the transmembrane domain of ORF1. While alternative splicing serves as a mechanism to increase the functional diversity of higher eukaryotes, its role in non-metazoan eukaryotes like Plasmodium , is poorly understood. The splicing pattern of post-transcriptional maebl products is conserved among different species of malaria suggesting it may have importance. The aim of this study is to determine if the attachment and invasion of anopheline mosquito salivary glands is dependent on the presence of one or both of the MAEBL isoforms. The experimental design will create mutant Plasmodium berghei parasites with maebl cDNA that will encode for only one specific isoform. My summer project was to create plasmid constructs for needed to create the mutant parasites lines by homologous recombination. Blood-stage maebl mRNA was isolated from P. berghei . cDNA was generated by RT-PCR, and inserted into a pGEM T-easy vector which contains a genetic sequence conferring resistance to ampicillin. The resulting plasmid was transformed into XL-Blue E.coli competent cells. Transformed were selected for by plating cells on agar containing ampicillin. DNA from transformed parasites was isolated. Open reading frames were determined by restriction enzyme digest/ gel electrophoresis and genomic sequencing. Orientation of cDNA within the vector was determined by restriction mapping and genomic sequencing. maebl cDNA and 3` untranslated sequences of P. berghei maebl of compatible orientation were ligated. Final constructs were cloned into plasmid pDEF-hDHFR.
Effects of Vitamin 1,25 (OH) 2 D 3 on Protein levels of Grim-19 and Stat3
Kellie K. Middleton. Belinda Byrne. JoEllen Welsh.
Breast cancer is the most common cancer among women, excluding nonmelanoma skin cancers. In addition, it is the second leading cause of cancer deaths in women today. Vitamin D in its metabolized form, 1 a ,25 (OH) 2 D 3 (1,25D), is a negative growth regulator of numerous cancer cells including breast cancer. Vitamin D 3 acts by binding its cognate receptor, the vitamin D receptor (VDR) and exerts negative growth-regulatory effects on breast cancer cells including cell cycle arrest and causes disruption of mitochondrial function to induce apoptotic death. The detailed mechanism of 1,25D -induced growth arrest and cell death remains unknown. In this study, investigation of apoptosis related proteins found by BD Biosciences Powerblot identified a number of proteins that may be involved in the Vitamin D 3 apoptotic cascade. Promising data was obtained from the BD Biosciences Powerblot on the mitochondrial protein Grim-19 (Gene associated with Retinoid-IFN induced Mortality). Previous studies have found that overexpression of this 16kDa protein enhances cell death in response to IFN-RA. Grim-19 forms aggregates at the cell’s perinuclear region with co-expressed anti-apoptotic protein Stat3 which belongs to a family of Signal Transducers and Activators of Transcription involved in anti-apoptotic activities. It’s phosphorylation at Tyr705 and Ser727 induces its dimerization and translocation into the nucleus. Grim-19 negatively regulates Stat3 by preventing its translocation into the nucleus and incidentally transcription of its target genes. In this study, the protein levels of Grim-19 and Stat 3 were measured in MCF-7 and a vitamin D resistant variant of MCF-7 (MCF-7 DR ) breast cancer cells in response to treatment with vitamin D 3 after 72, 120, and 144 hours. In the presence of 1,25D, both proteins were down regulated in MCF-7 and MCF-7 DR cells after 72 hours of treatment. These are novel findings that may lead to a better understanding of the mechanism of apoptosis induced by 1,25 D in breast cancer cells.
Characterization of Estrogen Receptor Signaling in a Novel Mammary Tumor Model System
Gennifer Goode, Meggan Valrance, and JoEllen Welsh
1,25-Dihydroxyvitamin D 3 (1,25D 3 ), the biologically active form of vitamin D 3 , is a steroid hormone that mediates effects on calcium homeostasis and cell growth through its nuclear receptor, the Vitamin D 3 receptor. 1,25D 3 has also been previously shown to act as an anti-estrogen, mainly by down-regulating the estrogen receptor (ER) protein. Estrogen (E 2 ) is a known survival factor for breast cancers. A novel breast cancer model system has been developed in the Welsh lab, consisting of two cell lines (WT145 and KO240) established from DMBA-induced mammary tumors in vitamin D 3 receptor wild-type (WT) and knock-out (KO) mice. In these studies we assessed the expression and activity of both ER isoforms (a and ß) the WT145 and KO240 cell lines. ER activity assessed in estrogen response element (ERE)-reporter gene assay, was increased significantly in both cell lines following E 2 treatment. Treatment with 1,25D 3 blocked estrogen-mediated ERE activation in WT145, but not KO240, cells. E 2 increased growth of KO240 cultures, which was blocked by the anti-estrogen tamoxifen (Tam). However, no growth increase was observed in WT145 cultures following E 2 treatment. To determine whether differential biological responses could be linked to differential expression of ER isoforms, Western blots with ERa and ERß specific antibodies were conducted. Both WT145 and KO240 cells express ERß , but neither cell line expresses ERa under the culture conditions used. Our results suggest that ERß is transcriptionally active in both cell lines, but is only coupled to induce growth regulatory pathways in KO240 cells. Further more, our data indicates that 1,25D 3 requires its nuclear receptor to act as an anti-estrogen.
A Study of Anopheles gambiae Opsins In Drosophila
DeAndrea Fedrick, Dr. Michelle Whaley and Dr. Joseph O’Tousa
Opsins are visual pigments that absorb light and start the phototransduction cascade. In Drosophila there are six well-characterized opsins. Anopheles gambiae has eleven opsins that have never been studied or characterized. The purpose of this project is to study the eleven opsins of Anopheles gambiae in the photoreceptor cells of Drosophila. Once the opsins are expressed in the photoreceptor cells, the spectral sensitivities will be tested along with comparative studies of Drosophila opsins and Anopheles opsins. Opsin seven was the only opsin that could be obtained for the study to date. Opsin seven was cloned into the P-element plasmid, pGasper, containing the nina E promoter. Drosophila embryos were collected every 15-30 minutes and were injected with the plasmid. The embryos were then incubated for 22-24 hours. 120 Larvae were collected and placed on fresh food. 82 G0 flies were then mated to a white balancer stock to map the insert. 27 G1 progeny have been analyzed thus far, but no transformants have been obtained. The study continues to look for transformants and then test the spectral sensitivities and expression pattern of opsin 7 in fly photoreceptors. The other ten Anopheles gambiae opsins will be analyzed as soon as they are obtained from TIGR (The Institute of Genomic Research).
Silencing Arfaptin 2
Bradley G. Changstrom , Holly Hoover and Crislyn D’Souza-Schorey
Department of Biological Sciences, University of Notre Dame
Huntington’s disease (HD) is an inherited autosomal dominant neurodegenerative disorder. HD affects approximately 1 in every 10,000 individuals. HD pathogenesis is characterized by gradual loss of coordination, cognitive and behavioral disturbances and eventual death. The disease is caused by an expanded glutamine (polyQ) repeat at the amino-terminus of the huntingtin protein. Nuclear inclusions and cytoplasmic protein aggregates are characteristic abnormalities that are associated with cellular toxicity induced by mutant huntingtin. Previous research from our laboratory has demonstrated that arfaptin 2 localizes to mutant huntingtin aggregates and is involved in regulating mutant huntingtin protein aggregation by inhibiting the proteosome.
Short-interfering RNA (siRNA) has been shown to be an effective post-transcriptional means of suppressing gene expression through RNA interference. This highly regulated enzyme-mediated process functions by targeting messenger RNA and results in cleavage of the target mRNA. The goal of this project was to determine if siRNA would be an effective strategy to suppress expression of arfaptin 2 in cultured neuronal cells.
Two rational 21 nucleotide target sequences were chosen through sequence analysis of the arfaptin 2 gene using siRNA analysis software. Subsequently, these target sequences were cloned into two distinct siRNA vectors. Western blot analysis shows that these generated siRNA are an effective means of arfaptin 2 gene silencing. Further siRNA studies will help elucidate the role of arfaptin 2 in HD pathogenesis by knockdown of endogenous arfaptin 2 levels.
Characterization of growth phenotypes in select fast-growing strains of Plasmodium falciparum
Malaria kills approximately 2 million people worldwide each year. It is caused by four species of the protozoan parasite Plasmodium , of which Plasmodium falciparum is the most virulent. Past studies on Plasmodium have linked virulence to parasite growth rate, indicating that more virulent parasites have higher proliferation rates 1 . Therefore, increased understanding of factors controlling parasitic growth may ultimately lead to methods of diminishing disease severity. In order to examine parasitic growth in human erythrocytes, the overall proliferation can be broken down into specific growth phenotypes corresponding to sections of the erythrocytic life cycle, such as cycle time, average number of merozoites per schizont, and invasion efficiency. In this study, these phenotypes are used in concert with a direct smear-based growth assay and a hypoxanthine incorporation assay to characterize the growth of four progeny and two isolate strains of P. falciparum . The progeny were selected from a group of 35 progeny resulting from a cross between two P. falciparum strains HB3 and Dd2. The two isolates Indo and FCB were collected in Southeast Asia. The exploration of parasitic growth leads to such questions as are there significant, quantifiable differences in growth rate between the strains? Which part of the life cycle causes a parasite to grow faster? Are faster-growing parasites less robust? This characterization of growth illustrates a significant difference in growth rates among parasite strains and proposes possible causes of these differences. In general, the isolate strains appear to have higher growth rates than the progeny, but the ranking of the parasites differ according to the method of growth measurement used.
The Effect of Pathogenic Mycobacteria on Macrophage Signaling
Upon infection mycobacteria engage host cell receptors which initiate intracellular signaling. The macrophage signaling proteins p38 and extracellular signal-regulated kinases 1 and 2 (ERK 1/2) of the mitogen–activated protein kinase (MAPK) pathways are activated during and infection resulting in the production of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-a) as well as other factors important for host immunity. Interestingly, we have previously found that MAPK activity is significantly diminished in macrophages following infection with pathogenic Mycobacterium avium strains relative to infection with non-pathogenic M. smegmatis . Additionally, macrophages infected with M. avium versus non-pathogenic mycobacteria produce significantly lower amounts of TNF-a. The mechanism through which M. avium modulates the macrophage response remains poorly understood. In these set of experiments we addressed the question of whether M. avium suppresses a macrophage response or fails to properly activate the macrophage. To characterize the macrophage response we looked MAPK activation and TNF-a production in M. avium infected macrophages which were subjected to a second infection with M. smegmatis or treated with the potent macrophage simulator lipopolysaccharide (LPS). Our studies showed that M. avium infection does not inhibit the M. smegmatis initiated activation of the MAPK or TNF-a production. Similarly, M. avium infected macrophages treated with LPS showed comparable stimulation relative to macrophages treated with LPS alone. Therefore, our data indicates that M. avium does not illicit a suppression of the macrophage response to subsequent stimuli. Instead, this suggests that pathogenic M. avium fails to activate the macrophage signaling necessary for a proinflamatory response.
Population Genetics of Anopheles funestus Giles in Sub-Saharan Africa
Kendan Jones-Isaac, Andy Michel, and Dr. Nora Besansky
In sub Saharan Africa three vector species of anopheline mosquitoes are responsible for the vast majority of human infections; Anopheles funestus, A. gambiae, and A. arabiensis . Of these species, A. funestus is possibly the most important as far as transmission during the dry season. However, despite its importance as a malarial vector , relatively little information on the population genetic structure of A. funestus is known. In the Besansky lab, work is being done to better understand this structure. Through analysis of microsatellite loci as well as a mitochondrial gene, an image of the population structure of A. funestus in the West African nation of Burkina Faso is being shaped. Preliminary analysis has indicated a possible speciation event within sympatric A. funestus populations. My experiments will provide a closer look at population structure of A. funestus between individual villages ranging from 50-100kms, using sequences from the mtDNA gene ND5 . The data collected will provide insights into not only populations within Burkina Faso but also throughout Africa.
Characterization of Antibody 3 Against the Cytoskeleton of Toxoplasma gondii
Cynthia Chaghouri
Dr. Kristin Hager
Toxoplasmosis, an infection caused by a single-celled parasite Toxoplasma gondii, causes neurological birth defects in newborns and severe infection is immunosuppressed individuals, such as patients with AIDS.
The COP1, a protein complex, trafficks proteins between the Golgi and the ER and within the Golgi compartment of Toxoplasma gondii . The beta-COP is a subunit of COP1 complex; Antibody 3 is an antibody generated against the b -COP peptide, but previous experiments suggested that Antibody 3 does not recognize the b -COP. Previous experiments have also depicted that the staining patterns of the Inner Membrane Complex (IMC), which recognizes IMC1 protein, appear to have similar staining patterns to that of Antibody 3. Therefore, there are three parts to this experiment: 1) Confirm that Antibody 3 does not recognize b -COP. 2) Confirm that Antibody 3 is not the same as IMC. 3) Determine what protein Antibody 3 recognizes, since it does not follow the expected staining pattern of b -COP.
To acquire these results, Immunofluorescence Assays (IFA), Immunoprecipitations (IP), and Western blot analysis were performed. IFA's were conducted to confirm that Antibody 3 did not recognize b -COP or any of the known IMC1 proteins. IP's and Western blot analysis are techniques used to separate and detect the target protein and to acquire the protein's molecular weight. Using these techniques, promising bands corresponding to proteins of interest were obtained. The candidate bands from the gels were then sent for sequencing to determine the protein that is being recognized by Antibody 3.
This protein, which Antibody 3 recognizes, may play a role in the cell division of Toxoplasma gondii , since it is predominantly present in dividing cells. Determining what protein is involved, may allow for the development of drugs that could prevent the cells from dividing and thus the spread of Toxoplasmosis.