Faculty Bibliography

Injection of Plasmodium salivary gland sporozoites into the vertebrate host by Anopheles mosquitoes initiates malaria infection. Sporozoites develop within oocysts in the mosquito midgut and then enter and mature in the salivary glands. Although morphologically similar, oocyst sporozoites and salivary gland sporozoites differ strikingly in their infectivity to the mammalian host, ability to elicit protective immune responses, and cell motility. Here, we show that differential gene expression coincides with these dramatic phenotypic differences. Using suppression subtractive cDNA hybridization we identified highly up-regulated mRNAs transcribed from 30 distinct genes in salivary gland sporozoites. Of those genes, 29 are not significantly expressed in the parasite's blood stages. The most frequently recovered transcript encodes a protein kinase. Developmental up-regulation of specific mRNAs in the infectious transmission stage of Plasmodium indicates that their translation products may have unique roles in hepatocyte infection and/or development of liver stages

Children and adolescents are increasingly exposed to psychostimulants, either illicitly or for the treatment of common neuropsychiatric conditions, such as attention deficit disorder with and without hyperactivity. Despite the widespread use of psychomotor stimulants in younger age groups, little is known regarding the chronic molecular neuroadaptive responses to these agents in the immature brain. Here we demonstrate that, after chronic administration of the psychostimulants cocaine and amphetamine, the transcription factor DeltaFosB is upregulated in the nucleus accumbens of periadolescent mice but not in post-weanling or adult mice. Induction of DeltaFosB also occurs exclusively in the caudate putamen of periadolescent mice after amphetamine administration. These results demonstrate the unique plasticity in the adolescent brain of a critical molecule that regulates psychostimulant action and suggest that these neuroadaptive changes may be involved in the mediation of enhanced addictive tendencies in the adolescent relative to the adult

Mice with fibroblast-specific expression of TAP-1 were generated by expressing the TAP-1 transgene under the control of the fibroblast-specific protein (FSP) 1 promoter/enhancer on TAP-1-deficient background. MHC class I expression in primary fibroblast cultures isolated from the resulting strain mimicked that of wild-type counterparts. MHC class I was detected in both types of fibroblasts following treatment with IFN-alphabeta. Positive selection of CD4(-)CD8(+) thymocytes was observed in neither adult nor fetal/neonatal thymus of transgenic mice. IFN-alphabeta-induced expression of MHC class I rescued positive selection of CD4(-)CD8(+) T cells in fetal thymic organ cultures, but not in adult mice. Contrary to previous suggestions, our results indicate a limited role of fibroblasts in promoting positive selection. In addition, the results suggest that positive selection may occur by a different mechanism in fetal vs adult thymus

Crry is a rodent membrane-bound inhibitor of complement activation and is a structural and functional analog of the human complement inhibitors decay-accelerating factor and membrane cofactor protein. We found previously that expression of rat Crry on a human tumor cell line enhances tumorigenicity in nude rats. In this study, we investigated the effect that rat Crry expressed on tumor cells has on rat cell-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC). The expression of rat Crry on the surface of different human tumor cell lines inhibited ADCC mediated by rat natural killer (NK) cells. C3 opsonization is known to enhance NK cell-mediated cytolysis, and a potential mechanism for Crry-mediated inhibition of NK cell lysis is through Crry modulation of C3 deposition on target cells. However, the transfection of tumor cell lines with Crry enhanced their resistance to NK cell-mediated lysis in the absence of exogenous complement. The resistance of Crry-expressing tumor cells to NK cell-mediated ADCC could be reversed by treatment with anti-Crry F(ab)(2). In addition, anti-Crry F(ab)(2) enhanced the susceptibility of 13762 rat mammary adenocarcinoma cells (that endogenously express Crry) to ADCC mediated by allogeneic rat NK cells in the absence of added complement. We found no evidence that rat NK cells were a source of complement for target cell deposition during the in vitro cytolysis assay. These data suggest a novel function for rat Crry in tumor immune surveillance that may be unrelated to complement inhibition

Chondrocyte proliferation is important for skeletal development and growth, but the mechanisms regulating it are not completely clear. Previously, we showed that syndecan-3, a cell surface heparan sulfate proteoglycan, is expressed by proliferating chondrocytes in vivo and that proliferation of cultured chondrocytes in vitro is sensitive to heparitinase treatment. To further establish the link between syndecan-3 and chondrocyte proliferation, additional studies were carried out in vivo and in vitro. We found that the topographical location of proliferating chondrocytes in developing chick long bones changes with increasing embryonic age and that syndecan-3 gene expression changes in a comparable manner. For in vitro analysis, mitotically quiescent chondrocytes were exposed to increasing amounts of fibroblast growth factor-2 (FGF-2). Proliferation was stimulated by as much as 8-10-fold within 24 h; strikingly, this stimulation was significantly prevented when the cells were treated with both fibroblast growth factor-2 (FGF-2) and antibodies against syndecan-3 core protein. This neutralizing effect was dose-dependent and elicited a maximum of 50-60% inhibition. To establish specificity of neutralizing effect, cultured chondrocytes were exposed to FGF-2, insulin-like growth factor-1, or parathyroid hormone, all known mitogens for chondrocytes. The syndecan-3 antibodies interfered only with FGF-2 mitogenic action, but not that of insulin-like growth factor-1 or parathyroid hormone. Protein cross-linking experiments indicated that syndecan-3 is present in monomeric, dimeric, and oligomeric forms on the chondrocyte surface. In addition, molecular modeling indicated that contiguous syndecan-3 molecules might form stable complexes by parallel pairing of beta-sheet segments within the ectodomain of the core protein. In conclusion, the results suggest that syndecan-3 is a direct and selective regulator of the mitotic behavior of chondrocytes and its role may involve formation of dimeric/oligomeric structures on their cell surface

We have used an approach using 2-dimensional gel electrophoresis with mass spectrometry analysis combined with oligonucleotide chip hybridization for a comprehensive and quantitative study of the temporal patterns of protein and mRNA expression during myeloid development in the MPRO murine cell line. This global analysis detected 123 known proteins and 29 'new' proteins out of 220 protein spots identified by tandem mass spectroscopy, including proteins in 12 functional categories such as transcription factors and cytokines. Bioinformatic analysis of these proteins revealed clusters with functional importance to myeloid differentiation. Previous analyses have found that for a substantial number of genes the absolute amount of protein in the cell is not strongly correlated to the amount of mRNA. These conclusions were based on simultaneous measurement of mRNA and protein at just a single time point. Here, however, we are able to investigate the relationship between mRNA and protein in terms of simultaneous changes in their levels over multiple time points. This is the first time such a relationship has been studied, and we find that it gives a much stronger correlation, consistent with the hypothesis that a substantial proportion of protein change is a consequence of changed mRNA levels, rather than posttranscriptional effects. Cycloheximide inhibition also showed that most of the proteins detected by gel electrophoresis were relatively stable. Specific investigation of transcription factor mRNA representation showed considerable similarity to those of mature human neutrophils and highlighted several transcription factors and other functional nuclear proteins whose mRNA levels change prominently during MPRO differentiation but which have not been investigated previously in the context of myeloid development. Data are available online at http://bioinfo.mbb.yale.edu/expression/myelopoiesis

OBJECTIVE: To explore the inhibitory effect of human endostatin gene mediated by retroviral vector on the growth of human liver carcinoma. METHODS: A recombinant retroviral plasmid containing human endostatin gene and signal peptide was engineered and transferred into PA317 cells to produce retrovirus. Human liver carcinoma cells (SMMC7721) were infected with the above retrovirus to build a stable endostatin-transfected liver carcinoma cell line (SMMC-endo). The control liver carcinoma cell line (SMMC-pLncx) was developed in a similar way except that the plasmid was replaced by an empty retroviral vector. Immunohistochemistry and Western blot were used to test the expression and secretion of human endostatin. The biological activity of the expressed human endostatin was assessed by endothelial cell proliferation assay. The growth rates of SMMC-endo and control SMMC-pLncx cells in vivo and in vitro were also observed. RESULTS: The expression and secretion of human endostatin by endostatin-transfected SMMC-endo cells were confirmed by immunohistochemistry and Western blot. Compared with the control group, concentrated supernatant of SMMC-endo cells remarkably inhibited the proliferation of human umbilical vein endothelial cells by 48%, significantly higher than the inhibition by the control (10.2%; P < 0.01). The endostatin-transfected SMMC-endo cells had similar in vitro growth rates to SMMC-pLncx cells. The in vivo experiment showed that the growth rate of SMMC-endo cells was slowed. Only in 3 out of 5 mice were tumors formed and flank tumors of SMMC-endo cells were 94.5% smaller than those of control cells 22 days after inoculation into nude mice (P < 0.001). CONCLUSIONS: Gene transfer of human endostatin mediated by retroviral vector is an effective form of cancer therapy

The Sonic hedgehog (Shh)-Gli signaling pathway regulates development of many organs, including teeth. We cloned a novel gene encoding a transcription factor that contains a zinc finger domain with highest homology to the Gli family of proteins (61-64% amino acid sequence identity) from incisor pulp. Consistent with this sequence conservation, gel mobility shift assays demonstrated that this new Gli homologous protein, GliH1, could bind previously characterized Gli DNA binding sites. Furthermore, transfection assays in dental pulp cells showed that whereas Gli1 induces a nearly 50-fold increase in activity of a luciferase reporter containing Gli DNA binding sites, coexpression of Gli1 with Gli3 and/or GliH1 results in inhibition of the Gli1-stimulated luciferase activity. In situ hybridization analysis of mouse embryos demonstrated that GliH1 expression is initiated later than the three Gli genes and has a more restricted expression pattern. GliH1 is first detected diffusely in the limb buds at 10.0 days post coitus and later is expressed in the branchial arches, craniofacial interface, ventral part of the tail, whisker follicles and hair, intervertebral discs, teeth, eyes and kidney. LacZ was inserted into the GliH1 allele in embryonic stem cells to produce mice lacking GliH1 function. While this produced indicator mice for GliH1-expression, analysis of mutant mice revealed no discernible phenotype or required function for GliH1. A search of the Celera Genomics and associated databases identified possible gene sequences encoding a zinc finger domain with approximately 90% homology to that of GliH1, indicating there is a family of GliH genes and raising the possibility of overlapping functions during development