Faculty Bibliography

Dendritic cells (DCs) are potent antigen-presenting cells with the potential for cancer immunotherapy. Adenoviral-mediated gene transfer is an attractive means to manipulate the immunostimulatory properties of DCs for therapeutic advantage. Because adenovirus induces DC maturation, we postulated that it would significantly alter their immune functions. Infected DCs markedly increased allogeneic and antigen-specific T-cell proliferation, and augmented natural killer cell lytic activity and IFN-gamma production. The enhanced effector cell stimulation by infected DCs was dependent on their secretion of interleukin 12. Immunization with infected DCs pulsed with tumor antigen protected against flank tumors in 78% of mice and induced a memory response. Antitumor immunity was dependent on both T cells and natural killer cells. Antigen-pulsed, mock-infected DCs were nonprotective. The findings that adenoviral vectors alone critically alter DC immune functions and antitumor properties have important implications for the design and interpretation of immunotherapy regimens using vector-based gene transfer to modulate immunity

GM-CSF is critical for dendritic cell (DC) survival and differentiation in vitro. To study its effect on DC development and function in vivo, we used a gene transfer vector to transiently overexpress GM-CSF in mice. We found that up to 24% of splenocytes became CD11c+ and the number of DC increased up to 260-fold to 3 x 10(8) cells. DC numbers remained substantially elevated even 75 days after treatment. The DC population was either CD8alpha+CD4- or CD8alpha-CD4- but not CD8alpha+CD4+ or CD8alpha-CD4+. This differs substantially from subsets recruited in normal or Flt3 ligand-treated mice or using GM-CSF protein injections. GM-CSF-recruited DC secreted extremely high levels of TNF-alpha compared with minimal amounts in DC from normal or Flt3 ligand-treated mice. Recruited DC also produced elevated levels of IL-6 but almost no IFN-gamma. GM-CSF DC had robust immune function compared with controls. They had an increased rate of Ag capture and caused greater allogeneic and Ag-specific T cell stimulation. Furthermore, GM-CSF-recruited DC increased NK cell lytic activity after coculture. The enhanced T cell and NK cell immunostimulation by GM-CSF DC was in part dependent on their secretion of TNF-alpha. Our findings show that GM-CSF can have an important role in DC development and recruitment in vivo and has potential application to immunotherapy in recruiting massive numbers of DC with enhanced ability to activate effector cells

Rat liver mitochondrial glycerophosphate acyltransferase (mtGAT) possesses 14 consensus sites for casein kinase II (CKII) phosphorylation. To study the functional relevance of phosphorylation to the activity of mtGAT, we treated isolated rat liver mitochondria with CKII and found that CKII stimulated mtGAT activity approximately 2-fold. Protein phosphatase-lambda treatment reversed the stimulation of mtGAT by CKII. Labeling of both solubilized and non-solubilized mitochondria with CKII and [gamma-32P]ATP resulted in a 32P-labeled protein of 85kDa, the molecular weight of mtGAT. Our findings suggest that CKII stimulates mtGAT activity by phosphorylation of the acyltransferase. The significance of this observation with respect to hormonal control of the enzyme is discussed.

Despite the importance of vascular smooth muscle cells in the regulation of blood vessel function, the molecular mechanisms governing their development and differentiation remain poorly understood. Using an in vitro system whereby a pluripotent neural crest cell line (MONC-1) can be induced to differentiate into smooth muscle cells, we isolated a cDNA fragment that was robustly induced during this differentiation process. Sequence analysis revealed high homology to a partial cDNA termed modulator recognition factor 2 (Mrf2). Because the full-length cDNA has not been reported, we cloned the full-length Mrf2 cDNA by cDNA library screening and 5' rapid amplification of cDNA ends and identified two isoforms of Mrf2 (alpha [3.0 kb] and beta [3.7 kb]) that differ in the N-terminus but share the DNA-binding domain. Protein homology analysis suggests that Mrf2 is a member of the AT-rich interaction domain family of transcription factors, which are known to be critically involved in the regulation of development and cellular differentiation. Mrf2alpha and Mrf2beta are highly induced during in vitro differentiation of MONC-1 cells into smooth muscle cells, and Mrf2alpha is expressed in adult mouse cardiac and vascular tissues. To define the function of Mrf2, we overexpressed both isoforms in 3T3 fibroblast cells and observed an induction of smooth muscle marker genes, including smooth muscle alpha-actin and smooth muscle 22alpha. Furthermore, Mrf2alpha and Mrf2beta retarded cellular proliferation. These data implicate Mrf2 as a novel regulator of smooth muscle cell differentiation and proliferation.

GAP-43 (neuromodulin) is a protein kinase C substrate that is abundant in developing and regenerating neurons. Thioester-linked palmitoylation at two cysteines near the GAP-43 N terminus has been implicated in directing membrane binding. Here, we use mass spectrometry to examine the stoichiometry of palmitoylation and the molecular identity of the fatty acid(s) attached to GAP-43 in vivo. GAP-43 expressed in either PC12 or COS-1 cells was acetylated at the N-terminal methionine. Approximately 35% of the N-terminal GAP-43 peptides were also modified by palmitate and/or stearate on Cys residues. Interestingly, a variety of acylated species was detected, in which one of the Cys residues was acylated by either palmitate or stearate, or both Cys residues were acylated by palmitates or stearates or a combination of palmitate and stearate. Depalmitoylation of membrane-bound GAP-43 did not release the protein from the membrane, implying that additional forces function to maintain membrane binding. Indeed, mutation of four basic residues within the N-terminal domain of GAP-43 dramatically reduced membrane localization of GAP-43 without affecting palmitoylation. These data reveal the heterogeneous nature of S-acylation in vivo and illustrate the power of mass spectrometry for identification of key regulatory protein modifications

Late generations of telomerase-null (TR(-/-)) mice exhibit progressive defects in highly proliferative tissues and organs and decreased fertility, ultimately leading to sterility. To determine effects of telomerase deficiency on germ cells, we investigated the cleavage and preimplantation development of embryos derived from both in vivo and in vitro fertilization of TR(-/-) or wild-type (TR(+/+)) sperm with either TR(-/-) or TR(+/+) oocytes. Consistently, fertilization of TR(-/-) oocytes with either TR(+/+) or TR(-/-) sperm, and TR(-/-) sperm with TR(+/+) oocytes, resulted in aberrant cleavage and development, in contrast to the normal cleavage and development of TR(+/+) oocytes fertilized by TR(+/+) sperm. Many (>50%) of the fertilized TR(-/-) eggs developed only one pronucleus, coincident with increased incidence of cytofragmentation, in contrast to the normal formation of two pronuclei and equal cleavage of wild-type embryos. These results suggest that both TR(-/-) sperm and oocytes contribute to defective fertilization and cleavage. We further found that a subset (7-9%) of telomeres was undetectable at the ends of some metaphase I chromosomes from TR(-/-) spermatocytes and oocytes, indicating that meiotic germ cells lacking telomerase ultimately resulted in telomere shortening and loss. Dysfunction of meiotic telomeres may contribute to aberrant fertilization of gametes and lead to abnormal cleavage of embryos, implying an important role of functional telomeres for germ cells undergoing fertilization and early cleavage development

Lytic infection by polyomavirus leads to elevated levels of p53 and induction of p53 target genes p21Cip1/WAF1 (p21) and BAX. This is seen both in polyomavirus-infected primary mouse cell cultures and in kidney tissue of infected mice. Stabilization of p53 and induction of a p53 response are accompanied by phosphorylation of p53 on serine 18, mimicking a DNA damage response. Stabilization of p53 does not depend on p19Arf interaction with mdm2. Cells infected by a mutant virus defective in binding pRb and in inducing G(1)-to-S progression show a greatly diminished p53 response. However, cells infected by wild-type virus and blocked from entering S phase by addition of mimosine still show a p53 response. These results suggest a role of E2F target genes in inducing a p53 response. Polyomavirus large T antigen coprecipitates with p53 phosphorylated on serine 18 and also with p21Cip1/WAF1. Implications of these and other findings on possible mechanisms of induction and override of p53 functions during productive infection by polyomavirus are discussed.

Ephrin/Eph signalling is crucial for axonal pathfinding in vertebrates and invertebrates. We identified the Drosophila ephrin orthologue, Dephrin, and describe for the first time the role of ephrin/Eph signalling in the embryonic central nervous system (CNS). Dephrin is a transmembrane ephrin with a unique N terminus and an ephrinB-like cytoplasmic tail. Dephrin binds and interacts with DEph, the Drosophila Eph-like receptor, and Dephrin and DEph are confined to different neuronal compartments. Loss of Dephrin or DEph causes the abberant exit of interneuronal axons from the CNS, whereas ectopic expression of Dephrin halts axonal growth. We propose that the longitudinal tracts in the Drosophila CNS are moulded by a repulsive outer border of Dephrin expression.

OBJECTIVE: Corticotropin-releasing hormone deficient mice (CRH-KO) serve as an interesting model to understand the role of CRH in the regulation of adrenomedullary system. The aim of this study was to compare tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) on the levels of gene expression and protein in adrenal medulla of CRH-KO mice, their CRH (+/+) mates and Sprague-Dawley (SD) rats. METHODS: Levels of TH and PNMT mRNA were determined by reverse transcription with subsequent polymerase chain reaction (RT-PCR) and quantified relatively to the housekeeper glyceraldehyde-3-phosphate dehydrogenase. The amount of TH and PNMT protein was determined by Western blot analysis and visualized by enhanced chemiluminiscence. RESULTS: We detected a clear signal of 645 bp for TH mRNA and of 260 bp for PNMT mRNA in adrenal medulla of rats and CRH (+/+) mice, with higher concentration of TH and PNMT mRNA in rat adrenal medulla. Subsequently, TH and PNMT immunoprotein was measured and we found significantly higher amount of TH and also PNMT protein in the rat compared to CRH (+/+) mice. On the other hand, the amount of TH and PNMT immunoprotein in adrenal medulla of CRH-KO mice was significantly lower compared to CRH (+/+) mice. CONCLUSIONS: Our results indicate the lower production of adrenomedullary TH and PNMT protein in CRH (+/+) mice compared to rats, which reflects the lower gene expression of these enzymes in adrenal medulla of mice. We also demonstrated the differences in TH and PNMT protein levels between CRH (+/+) and CRH-KO (-/-) mice.