Cerebral palsy (CP) is a common neurologic disorder in children. It results from damage to the brain occurring before, during, or shortly after birth. A primary deficit in CP is neuromuscular dysfunction, and we have shown that at least some children with CP have abnormally distributed acetylcholine receptors (AChRs) relative to the functional neuromuscular junction (NMJ) as defined by acetylcholine esterase (AChEase) immunoreactivity. In the present study, the relationship between the distribution of AChRs outside the NMJ and the degree of motor-system affect of the CP child was investigated. Biopsies of lower limb muscles (Vastus, Gracilus and Gastrocnemius) were double stained in order to detect AChR and AChEase. Image analysis using a customized software macro was performed, and the Spread of AChR staining outside NMJs was calculated using biopsies of 59 children. Comparisons between ambulatory (n=21) and non-ambulatory (n=38) children indicated that non-ambulatory children had higher median Spread values (p=0.025) and a higher ratio of abnormal (Spread > mean + 2 x S.D. for normal NMJs) to total NMJs than ambulatory children (p=0.023). These results suggest that the level of affect in children with CP is associated with the presence of structurally pathological NMJs. This project was supported by the Nemours Foundation.

CRASH syndrome (Corpus callosum hypoplasia, Retardation, Adducted thumbs, Spastic paraplesia, and Hydrocephalus), a congenital birth defect in humans, is responsible for a spectrum of debilitating symptoms caused by the absence or misexpression of the cell adhesion molecule L1.  We believe that comparable abnormalities can be created in developing chick embryo brains by locally modifying expression of the structurally similar and functionally equivalent protein, NgCAM.  NgCAM will be attenuated by use of a 562 base pair antisense sequence that is complementary to the corresponding NgCAM coding sequence.  These sequences will be introduced into the chick brain via the RCASBP(A) replication-competent retroviral vector.  After incorporation into the host genome and expression, they will bind to the host’s NgCAM mRNA and will block its translation.  We hypothesize that this attenuation will cause disruption of normal neuronal development, thereby mimicking the effects of CRASH syndrome.  Effects are expected on migration of a subset of neurons and on axon extension and fasciculation.  In the end, the intention is to create a simpler and more easily manipulated model than the existing mouse models for the study of brain abnormalities in CRASH syndrome.  Supported by HHMI and NIH

The focus of our laboratory is to investigate the extracellular interactions that influence signaling pathways between cells.  Correct signaling and signal reception is vital for an organism to correctly develop.  Many diseases in humans have been linked to incorrect cell signaling, cancer being one example.  The information gathered by our research on Drosophila melanogaster can be applied to our knowledge of human cell signaling since many genes and cellular mechanisms are conserved throughout evolution.  Two examples of signaling pathways conserved throughout evolution and that are crucial to vertebrate development are the Wingless (Wg) and Hedgehog (Hh) signaling pathways.  These pathways are perfect as the focus of our study on cell signaling because they are both greatly influenced by the extracellular environment.   7H24, an ethylmethane sulfonate (EMS) mutation, was previously found to be linked to Wg and/or Hh signaling due to its maternal effect embryonic phenotype. The goal of my project is to map 7H24 to its corresponding gene through various genetic mapping techniques, then to determine the consequences of 7H24 on development through genetic and molecular characterization.  By analyzing the effect 7H24 has on Drosophila development, we can gain an understanding of the role the disrupted gene plays in Wg/Hh signaling.  This will help to shed light on the extracellular interactions and regulatory mechanisms required for correct signaling, as well as how disruptions in this signaling can lead to abnormal growth and development. Supported by VWR international and UDRF.

Calcium and Integrin Binding (CIB) protein, which interacts with the integrin alphaIIbbeta3, plays an important role in platelet spreading on immobilized fibrinogen. After screening a human mammary epithelial cDNA library in the yeast two-hybrid system using CIB as bait, a 247 amino acid polypeptide was isolated. This protein was stably expressed in NIH3T3 cells and a 2-3-fold increase in cell surface area was observed using immunofluorescence microscopy. We also observed an increased amount of stress fibers as visualized by phalloidin staining of F-actin. When CIB was overexpressed in these cells we found about a 40% decrease in cell surface area, suggesting that CIB inhibits the ability of this novel protein to induce cell spreading. Therefore, we named this protein CIB Regulated Adhesion and Spreading Stimulator-1 (CRASS-1). Since cell adhesion is a prerequisite for spreading, we asked whether CRASS-1 also effects cell adhesion. In adhesion assays performed with NIH3T3 cells on a fibronectin matrix, approximately a 2-fold increase in cell adhesion was observed in cells expressing CRASS-1 and this adhesion was decreased by 40% when CIB was expressed in these cells. Thus, our studies suggest that together with CIB, CRASS-1 may regulate cell adhesion and migration. KMM was funded by a Charles Peter White summer fellows grant. 

During the process of SV40 DNA replication initiation, four major proteins interact with each other to form an initiation complex that unwinds the origin of replication and begins the process of DNA synthesis.  First, large T antigen, the only viral protein required for DNA replication binds to the origin and forms a double hexamer.  This multifunctional protein must interact with topoisomerase I (topo), DNA pol¦Á/primase (DNA pol), and replication protein A (RPA) in order for DNA replication to initiate.  All three of these proteins have been shown to interact with T antigen in protein binding assays.  First, we determined the effects of each protein on initiating origin unwinding, and second, we investigated the form of circular DNA that serves best as a template in DNA replication.  These experiments were designed to shed light on the initiation of SV40 replication, one of the best known models to study a simplified version of the complicated process of mammalian DNA replication.  Previous data from origin unwinding assays have shown that when increasing amounts of DNA pol are added into in vitro replication conditions containing T and topo, more origin DNA becomes unwound that when DNA pol is absent.  DNA replication assays are now being performed with small circular double stranded DNAs to mimic the conditions used in the DNA unwinding assays.  In this way, we hope to discover the DNA template that serves best in DNA replication. DM supported by the HHMI Undergraduate Science Education Program.

The heparin/heparan sulfate (Hp/HS) interacting protein (HIP/RPL29) is identical to a ribosomal protein of the large subunit of ribosomes: L29.  It has been previously shown that HIP/RPL29 is expressed throughout growth plate cartilage.  The expression pattern shows that HIP/RPL29 expression is tightly down-regulated during chondrocyte terminal differentiation.  Our hypothesis is that a decrease in HIP/RPL29 expression is associated with a progression towards a late chondrocytic state.  To develop an in vitro system to study the importance of HIP/RPL29 during early chondrogenesis, we analyzed the chondrogenic potential of the totipotent mouse embryonic stem (ES) cell line, AB-1.  This ES cell line was cultured into embryoid bodies (EB) using the “hanging drop” method followed by suspension culture in the presence of bone morphogenetic protein 2 (BMP-2) (2-100 ng/ml).  EBs were plated onto plastic dishes coated or not with a domain of perlecan.  The chondrogenic nature of the plated EBs was determined after 8-9 days of culture on the basis of positive Alcian Blue staining.  Our results show that under these experimental conditions AB-1 ES cells not only differentiate into various morphological structures including cartilage-like aggregates, but also into cardiomyocyte contractile fibers.  Future work will optimize conditions to compare the chondrogenic potential of HIP/RPL29 targeted ES cells versus the parental wild type line.  (This work was supported by NIH grant HD25235 to D.D.C.)  Additional funding was received from the Science and Engineering Scholars Program.

The mode of adaptation to metabolic acidosis was studied in a primary cell culture of chick (Gallus domesticus) proximal tubule (PT) cells.  This was performed under a novel experimental model that allows for polarization of the PT-cells in culture.  Preliminary data indicates that cells under metabolic acidosis exhibit an increased rate of ammonia excretion.  This is accomplished mainly through the catabolism of L-glutamine.   A product of this catabolism is NH₄⁺ which is primarily transported across the apical side of chick PT-cells via a Na⁺/H⁺ exchanger. In mammals this exchanger is inhibited by parathyroid hormone (PTH), leading to decreased renal bicarbonate reabsorption. The effect of PTH on chick PT-cells is not well known. Therefore a comparative analysis was done with opossum kidney (OK) cells, an established mammalian cell line.  A three day protocol was used for both cell types with either acidic or normal pH media, with or without PTH.  Samples were taken from the apical and basolateral sides for analysis of ammonia. With the addition of PTH the OK cells did show a net decrease in NH₄⁺ production.  Chick PT-cell responses were not as well defined, which could be due to the use of alternate transporters. AM funded by HHMI 

Astrocytes maintain synapse function in the brain by taking up the neurotransmitter glutamate and converting it to glutamine.  Both of these reactions are highly energy-demanding and will occur too slowly if ATP is not rapidly regenerated.  In astrocytes and neurons, ATP is rapidly regenerated by the brain creatine kinase (CKB) enzyme.  For brain cells to produce sufficient amounts of CKB enzyme they must regulate transcription of the CKB gene.  Previous transfection experiments have shown that transcription factor AP2alpha can greatly induce transcription of CKB in U87-MG glioblastoma cells, an attractive model cell system for astrocytes.  Additional previous experiments showed that untransfected U87-MG do not contain the AP2alpha protein but rather an AP2 protein resembling AP2delta.  Our transfection experiments have shown that AP2delta can induce transcription of CKB but not as much as AP2alpha.  In addition, we have investigated whether the coactivator protein PC4 cooperates with AP2delta to elicit maximal induction of CKB transcription.  Understanding how AP2delta and PC4 cooperate in regulating CKB transcription will reveal how the levels of the CKB enzyme are maintained to permit proper ATP regeneration in astrocytes and possibly neurons.  DN and BG funded by the Howard Hughes Medical Institute.

Prostate cancer cells tend to metastasize to bone, where they proliferate with  the stimulation of local growth factors.  Voltage-sensitive calcium channels (VSCCs) located in the plasma membrane are the primary passageway for extracellular calcium to move into cells and act as a second messenger. Since bone is calcium rich, these channels are potential contributors to the growth  of prostate cancer in bone.  This study aimed to identify the major calcium channel subtypes found in the C4-2B4 prostate cancer cell line in order to find  targets that are not common in normal bone cells. C4-2B4 cells were cultured, total RNA was extracted, and RT-PCR techniques were used to test for expression of the various channel subtypes of the pore-forming alpha1 subunit as well as  the auxiliary beta, alpha2-delta, and gamma subunits. Immunohistochemical staining was then conducted for the L-type alpha1C and alpha1D subunits using specific rabbit derived antibodies to localize these channel subtypes. An Ion  Channel Superarray™ also was conducted using C4-2B4 cells to examine channel expression in response to treatment with 1alpha,25-Dihydroxyvitamin D₃, a known growth inhibitor of prostate cancer. Based upon our results, we concluded that  in C4-2B4 cells, the alpha1D subunit seems to be more highly expressed than alpha1C, which is the major channel found in osteoblasts and other bone cells. This study was funded by the Howard Hughes Medical Institute. 

Pseudoexfoliation Syndrome (PSX), a disorder diagnosed by fibrillar extracellular matrix (ECM) deposition in ocular tissue, is a major cause of open-angle glaucoma in addition to ocular surgery complications.  The buildup of ECM observed has led to the hypothesis that PSX is related to the inappropriate breakdown of extracellular matrix and basement membranes in the eye.  Since aqueous humor is the fluid bathing the ocular tissues affected by PSX, SELDI mass spec profiling of aqueous humor proteins was used to identify proteins contributing to PSX pathogenesis.  Initially, the NP20 ProteinArray, a chip whose active spots contain silicon oxide allowing the binding of hydrophilic proteins, was used.  The NP20 protein chip spectrums have contained a number of reproducible mass peaks, though the low accuracy of the specific mass values has necessitated the use of chromatographic surface chips that allow the binding of smaller subsets of proteins.  In parallel, a candidate gene approach has identified the presence of Cathepsin B, a protein associated with the inappropriate breakdown of extracellular matrix in a number of diseases, in both normal and PSX aqueous humor through Western blotting.  Sources of funding include HHMI, Undergraduate research, and PHS.

HIP/RPL29 is a heparin/heparan sulfate (Hp/HS) binding protein identical to ribosomal protein L29. Previous studies indicated that HIP/RPL29 is down regulated during the terminal phase of chondrogenesis and that partial reduction of HIP/RPL29 using an in vitro ribozyme knock-down approach accelerates differentiation of C3H/10T1/2 cells into cartilage-like cells. Because cartilage serves as a template for endochondral bone formation, we postulate that a strict regulation of HIP/RPL29 expression is essential for normal bone growth.  Our hypotheses are: 1) HIP/RPL29 down-regulation is essential for chondrocyte terminal differentiation and reciprocally, 2) HIP/RPL29 overexpression prevents terminal chondrocyte differentiation.  In this study, we microinjected the active HIP/RPL29 ribozyme into one-cell stage fertilized embryos and observed subsequent in vitro embryonic development. Future studies will consist of transferring embryos microinjected with HIP/RPL29 ribozyme into pseudopregnant surrogate mothers to study the effect of HIP/RPL29 down-regulation on in vivo bone development.  In parallel we created tools to create stable cell lines overexpressing HIP/RPL29 and cloned the entire sequence encoding for HIP/RPL29 downstream of the strong CMV promoter into an expression vector including a positive neomycin phophotransferase selection cassette to generate stable transfectants using G-418.  Optimal concentrations of G-418 and transfection conditions were established for the chondrocytic cell line ATDC5.  Collectively, these studies will provide valuable tools to study the importance of HIP/RPL29 regulation during in vitro and in vivo chondrogenesis.

Cell adhesion molecules of the Ig superfamily play an important role in embryonic development. We have recently shown that JAM-1, a member of this family, is involved in endothelial cell adhesion and migration leading to angiogenesis. We hypothesized that JAM-1 may also play a role in vasculogenesis; however, embryonic expression of JAM-1 is not well characterized. In order to understand the role of JAM-1 in vascular development/function, we studied the expression of JAM-1 during early embryonic development by generating transgenic mice in which a lacZi gene was knocked into the JAM-1 gene. JAM-1 gene expression was determined using histochemical staining for beta-galactosidase in mouse embryos ranging from 9.5 to 12.5 days post coitum (dpc). As expected, JAM-1 is expressed at sites of vasculogenesis, namely, intersomitic vessels, as early as 9.5 dpc. Expression in blood vessels is continuous through 12.5 dpc where staining is clearly visible in both the head and tail of the embryo. JAM-1 is also detected in the otic and olfactory placodes, which form the epithelial linings of the inner ear and nose, and the epithelia of the developing kidneys and lungs, which undergo branching morphogenesis. Thus, JAM-1 may function in the epithelial development of certain tissues. JJP supported by HHMI Undergraduate Science Education Program.

Adipocytes are known to be important regulators of fatty acid homeostasis.   The Serial Analysis of Gene Expression (SAGE) library of 3T3-L1 adipocytes, constructed in our laboratory, has also implicated them in having an important role in cholesterol homeostasis.  Several genes involved in cholesterol transport, Cav, SREBP-1c, Abca1, and Abcg1, were highly expressed.  To determine relative changes in gene expression from 3T3-L1 fibroblasts to differentiated adipocytes, 3T3-L1 cells were harvested daily during differentiation, and RNA was extracted with trizol.  Relative levels of Cav, SREBP-1c, Abca1, and Abcg1, were determined by real-time RT-PCR, and all showed an increase in level, over TATA box-binding protein, during 3T3-L1 differentiation. AP funded by HHMI Undergraduate Science Education Program

The complex signal transduction pathways that lead to events such as blood platelet activation and, as a result of this process, hemastasis, involve several key molecules.  One such molecule, calcium- and integrin-binding protein (CIB), a 22-kDa polypeptide, is already known to be greatly involved in the platelet activation pathway.  Thus, it has been hypothesized that CIB, which is highly present in prostate cancer (PC3) cells, might also play a similar signaling role in these cells.  Several membrane-associated signaling molecules are abundant in cholesterol-rich lipid rafts, infolds of the plasma membrane usually characterized by a 22-kDa aptly named protein called caveolin.  In order to study CIB’s relationship with lipid rafts, or caveolae, a detergent extract of the cells was subjected to isopycnography to separate the lipid rafts from total cell extract.  After confirming its general presence in PC3 cells through Western Blot analysis, distribution of caveolin in PC3 cells was determined using the isopycnographically produced fractions correlating to lipid rafts of the cells.  Presence of CIB in the lipid raft fractions was determined through Western Blotting which showed that CIB was not present in these fractions.  Since both CIB and caveolin are not simultaneously localized in the “resting” PC3 cell, it is possible that after stimulation with an agonist, CIB may associate with lipid rafts.  Experiments to confirm this hypothesis are ongoing.  Funded in part by Charles Peter White Scholars Program.

Cell-to-cell interactions and cell to extracellular matrix protein interactions play an important role in incurable illnesses like cardiovascular disease and cancer.  We have previously identified a calcium and integrin binding protein (CIB) which is involved in cell migration and cell spreading.  Another protein was identified in our laboratory which is known to interact with CIB.  It was named the CIB Regulated Adhesion and Spreading Stimulator, CRASS-1, because of its involvement in cell spreading.  Human genome search revealed that there exits another protein with 90% similarity to CRASS-1.  We named this protein CRASS-2.  In order to determine the function of CRASS-2, NIH3T3 (mouse fibroblast) cells were transfected with CRASS-2 cDNA.  A cell adhesion assay performed with CRASS-2 or Vector transfected cells indicated that CRASS-2 induced cell adhesion on fibronectin.  Consistent with enhanced adhesion, immunofluorescence experiments exhibited CRASS-2 localization to the stress-fibers of the cell.  Effect of expression of CIB on CRASS-2-induced cell adhesion is ongoing.  By determining the function of CRASS-2 and its regulation by CIB, it will be possible to uncover and establish, more exhaustively, the functional roles and capabilities of both CRASS-2 and CIB.  This will allow us to further expand our knowledge and understanding of cell migration and platelet aggregation that can instigate diseases like cancer, myocardial infarction and stroke. SP funded by the McNair Scholars Program.

It has been well established that androgenic hormones are potent and obligatory survival factors in vivo for secretory epithelial cells of the prostate gland. This dependence becomes apparent in the dramatic wave of apoptosis that is rapidly induced in the prostate in response to the removal of circulating androgen by castration (5).  The major obstacle posed by advanced prostate carcinoma is androgen independence and bone metastasis.  It seems that prostate carcinoma cells growing in the bone develop a characteristic for no longer being dependent on circulating androgen. The adhesion of cancer cells to the bone extra-cellular matrix is mediated through integrins, which activate intracellular signaling pathways that influence the behavior of the cell. We are interested in FAK (focal adhesion kinase); which is believed, when activated, recruits phosphatidylinositol 3-kinase (PI3K) which can directly activate intracellular cell survival pathways. The cell survival function of the PI3K pathway is mediated in part, by Akt-dependent inactivation of proapoptotic proteins (5). Since prostate cancer cells preferentially metastasize to the bone, it is hypothesized that PC-3 cells, when grown on a bone matrix, will produce a higher signal of activated FAK and activated Akt, when compared to PC-3 cells grown on plastic and kidney matrices.  FP supported by the Howard Hughes Medical Institute. 

Communication between adipocytes and endothelial cells is essential for regulating the amount of free fatty acids allowed to transverse the endothelial layer.  Serial analysis of gene expression (SAGE) used to obtain gene expression profiles of 3T3 L1 adipocytes and preadipocytes suggested two genes potentially responsible for such communication, orosomucoid (Orm1) and angiopoietin-like 4 (angptl4) are expressed.  The concentration of message during differentiation of mesenchymal cells to adipocytes was measured using Real-Time RT-PCR.   Trizol was used to extract RNA from cells harvested daily for two weeks.  An ABI Prism 7000 with SYBR-green detection was used to quantify the relative concentrations of mRNA for the two genes.  After induction of differentiation the 3T3 L1 cells begin to accumulate lipid around day 3 and reach capacity around day 7.  Orm1 expression increased 30-fold and angptl4 expression increased 6-fold greater than the internal marker tata-box binding protein (tbp).   Orm1 and Angptl4 are both upregulated early during differentiation, around day 3.  Orm1 reaches its maximum expression at day 12, whereas angptl4 reaches maximum expression around day 3 and remains static throughout the rest of differentiation.  These results along with previously published data suggest that orm1 and angptl4  secretion by adipocytes may inhibit fatty acid uptake by adipose tissue. Funded by Charles Peter White.

This study was conducted to characterize the expression of a hyaluronidase-like gene member, Hyal5.  Primers were designed from the 3UTR of Hyal5 to ensure that only DNA encoding the mRNA would be amplified.  Immunocytochemistry and immunohistochemistry were performed to determine the cell types in which the protein is expressed.  However, the results for the protein expression are inconclusive.  This is partly due to the fact that a Western Blot revealed the primary antibody used is not specific: it binds to Hyal5, Hyalp1, and Spam1, three closely linked hyaluronidase family members.  A developmental RT-PCR experiment was performed on testis RNA from mice that were the following ages:  27 days, 25 days, 21 days, 18 days, and 12 days.  RT-PCR was also performed on CREM-knockout mice to determine if Hyal-5 has a CRE sequence in its promoter region in order to confirm bioinformatic studies of the gene. This study was supported by grants from the Howard Hughes Medical Institute and National Institutes of Health. 

The active metabolite of vitamin D, 1,25(OH)2D₃, acts through nuclear receptor-mediated and plasma membrane initiated mechanisms. In the rat intestinal epithelial cell line, IEC-6, a series of rapid events result from the binding of 1,25(OH) 2D₃ to its basolateral receptor including G-protein coupled activation of phospholipase C, increases in intracellular cyclic adenosine monophosphate (cAMP), and transcaltachia (Ca²⁺ transport). The novel non-isotopic assay that we utilized measures cAMP levels after  activation of adenylate cyclase in response to 1,25(OH)2D₃ treatment. This fluorescent cAMP assay (Bridge-It™) is based on the activity of cAMP receptor protein (CRP), a bacterial sequence-specific DNA binding protein. Fluorescence resonance energy transfer (FRET) between the fluorochromes introduced to these DNA molecules detects protein-dependent association of the two DNA fragments. The assay mixture of the Bridge-It™ fluorescence cAMP assay kit contains CRP and the two fluorochrome-labeled DNA half-sites and thus contains all components necessary to generate a fluorescence signal in the presence of cAMP. We used the Bridge-It™ assay to determine if 1,25(OH)2D₃ increased cAMP in IEC-6 cells via its membrane receptor. A standard curve was developed over the summer that will allow us to assess cAMP responses in IEC-6 cells that contain varying levels of a candidate 1,25(OH)2D₃ membrane receptor. JOU supported by the HHMI Undergraduate Science Education Program.

HYALP1, a member of the hyaluronidase family, is an expressed psuedogene (resulting from two deletions that cause premature termination, Csoka et.al., 1999) located on human chromosome 7.  The mouse homologue, Hyalp1, is a functional gene expressed in the testis along with two other closely linked hyaluronidases, Hyal5 and Spam1 (sperm adhesion molecule 1).  Spam1 is widely conserved and has at least three essential roles in mammalian fertilization. However, a recent study of mice with the null mutation revealed no loss of fertility, suggesting that some of the roles attributed to Spam1 may be shared by its closely linked family members, Hyal5 and Hyalp1.  The aim of this investigation was to characterize the expression of Hyalp1 in the mouse, specifically, to determine if it is present on sperm and in the epididymis, as is the case for Spam1.  To determine its possible presence on sperm, a developmental RT-PCR analysis was performed using RNA from testes of 12, 18, 21, and 25 day-old mice.  Caput and cauda epididymal tissues were similarly analyzed after sperm was removed. RT-PCR showed the presence of Hyalp1 in the adult testis. Attempts at the protein expression by immunohistochemistry gave inconclusive results, partly due to the ineffectiveness of the antibody.  These studies are continuing. KW funded by the BRIN Program. 

Three major proteins are involved in the elongation step of Simian Virus 40 DNA synthesis. PCNA, Replication factor C (RF-C), and Polymerase delta are required for efficient elongation of growing strands.  My initial project was to purify two of these proteins, RF-C and polymerase delta.  RF-C is a complex of five subunits and polymerase delta is comprised of two subunits.  The recombinant baculoviruses expressing these subunits were first tested by immunofluorescence.  The best combinations of virus and insect cells were used for the purification prep.  High 5 insect cells were infected with baculoviruses encoding each individual subunit of the protein.  For RF-C, one of the subunits was 6 His-tagged and had the ability to bind to Ni-agarose beads.  After binding, the Ni-agarose beads were washed and the protein was eluted using imidazole.  Polymerase ? was purified by using two chromatographic columns.  Protein preps were analyzed for concentration and subunit expression by SDS PAGE gels, dot blots, and western blots.  Now that all three proteins have been purified, the next step will be to perform and characterize the elongation reaction.  This project was sponsored by the Howard Hughes Medical Institute.