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PubMed2006

Wdr5, a WD-40 protein, regulates osteoblast differentiation during embryonic bone development.

Wdr5 accelerates osteoblast and chondrocyte differentiation in vitro, and is developmentally expressed in osteoblasts as well as in proliferating and hypertrophic chondrocytes. To investigate the role of Wdr5 during endochondral bone development, transgenic mice overexpressing Wdr5 under the control of the 2.3-kb fragment of the mouse alpha(1) I collagen promoter were generated. The transgene was specifically expressed in the osteoblasts of transgene positive mice and was absent in the growth plate. Histological analyses at embryonic day 14.5 demonstrated that the humeri of transgene positive embryos were longer than those isolated from wild-type littermates largely due to an expansion of the hypertrophic chondrocyte layer. Acceleration of osteoblast differentiation was observed with greater and more extensive expression of type I collagen and more extensive mineral deposition in the bone collar of transgene positive embryos. Acceleration of vascular invasion was also observed in transgene positive mice. Postnatal analyses of transgenic mice confirmed persistent acceleration of osteoblast differentiation. Targeted expression of Wdr5 to osteoblasts resulted in earlier activation of the canonical Wnt signaling pathway in the bone collar as well as in primary calvarial osteoblast cultures. In addition, overexpression of Wdr5 increased the expression of OPG, a target of the canonical Wnt signaling pathway. Overall, our findings suggest that Wdr5 accelerates osteoblast differentiation in association with activation of the canonical Wnt pathway.

Animals↗
PubMed2004

Genome-wide microarray analysis of TGFbeta signaling in the Drosophila brain.

BACKGROUND: Members of TGFbeta superfamily are found to play important roles in many cellular processes, such as proliferation, differentiation, development, apoptosis, and cancer. In Drosophila, there are seven ligands that function through combinations of three type I receptors and two type II receptors. These signals can be roughly grouped into two major TGFbeta pathways, the dpp/BMP and activin pathways, which signal primarily through thick veins (tkv) and baboon (babo). Few downstream targets are known for either pathway, especially targets expressed in the Drosophila brain. RESULTS: tkv and babo both affect the growth of tissues, but have varying effects on patterning. We have identified targets for the tkv and babo pathways by employing microarray techniques using activated forms of the receptors expressed in the brain. In these experiments, we compare the similarities of target genes of these two pathways in the brain. About 500 of 13,500 examined genes changed expression at 95% confidence level (P < 0.05). Twenty-seven genes are co-regulated 1.5 fold by both the tkv and babo pathways. These regulated genes cluster into various functional groups such as DNA/RNA binding, signal transducers, enzymes, transcription regulators, and neuronal regulators. RNAi knockdown experiments of homologs of several of these genes show abnormal growth regulation, suggesting these genes may execute the growth properties of TGFbeta. CONCLUSIONS: Our genomic-wide microarray analysis has revealed common targets for the tkv and babo pathways and provided new insights into downstream effectors of two distinct TGFbeta like pathways. Many of these genes are novel and several genes are implicated in growth control. Among the genes regulated by both pathways is ultraspiracle, which further connects TGFbeta with neuronal remodeling.

Animals↗
PubMed1999

Sexual behavior mutants revisited: molecular and cellular basis of Drosophila mating.

The study of Drosophila melanogaster by a combination of forward genetics with specific mutants, and reverse genetics, in which a given gene is expressed in an appropriate brain area to test its effect on behavior, provides a unique opportunity to explore the causal relationship between a particular gene, its function in the cell and the behavioral outcome at the organismic level. Enhanced male-to-male courtship has been shown to occur as a result of mutations in several different genes. For example, the Voila mutant exhibits intense GAL4 reporter expression in the tarsal gustatory sensilla, suggesting the importance of tapping by a male on the female abdomen with his forelegs. Feminization of parts of the antennal lobe and mushroom body by targeted expression of a female-determining gene transformer+ (tra+) drives the male to court other males. Mutations in the tra target gene fruitless (fru), which is expressed in the antennal lobe as well as the suboesophageal ganglion (the gustatory inputs are processed here), also induce homosexual courtship in males. These results suggest that sensory inputs mediated and/or processed by the tarsal receptors, suboesophageal ganglion, antennal lobe and mushroom body contribute to the regulation of male-female courtship. Mosaic analysis localized the neural center for male courtship behavior to the posterior dorsal brain, in which the sensory information processed by the aforementioned neural structures may be integrated. Another mosaic study mapped the neural center for female sexual behavior, as measured by her receptiveness to copulation, to the anterior dorsal brain. The issue as to how the mutations that reduce female sexual receptiveness, e.g. dissatisfaction (dsf), spinster (spin) and chaste (cht), affect the structure and/or function of this neural center deserves to be addressed urgently.

Animals↗
PubMed2005

The protein kinase C pathway acts through multiple transcription factors to repress gonadotropin-releasing hormone gene expression in hypothalamic GT1-7 neuronal cells.

The GnRH gene uses two well-defined regions to target expression to a small population of hypothalamic GnRH neurons: a 173-bp proximal promoter and a 300-bp enhancer localized at approximately -1800 to -1500 bp from the start site. Interaction of multiple factors with the GnRH enhancer and promoter is required to confer neuron-specific expression in vivo and in cells in culture. In addition, the expression of the GnRH gene is regulated by numerous neurotransmitters and hormones. Several of these effectors act through membrane receptors to trigger the protein kinase C pathway, and 12-O-tetradecanoyl phorbol-13-acetate (TPA), a modulator of this pathway, has been shown to suppress GnRH gene expression through the promoter. We find that TPA suppresses expression through the GnRH enhancer as well as the promoter. In the enhancer, an Oct-1 binding site, a Pbx/Prep binding site, Msx/Dlx binding sites, and a previously unidentified protein-binding element at -1793, all contribute to TPA suppression. TPA treatment leads to decreased binding of Oct-1 and Pbx1a/Prep to their sites. However, a complex formed by GT1-7 nuclear extracts on the -1793 site is not affected by TPA treatment. It is known that cooperative interaction among multiple factors is necessary for GnRH gene expression; thus, one mechanism by which TPA suppresses GnRH gene expression is to disengage some of these factors from their cis-regulatory elements.

Animals↗
PubMed1996

Chronic complement C3 gene expression in the CNS of transgenic mice with astrocyte-targeted interleukin-6 expression.

Both cytokines and complement are thought to play significant, but poorly understood roles, in the pathogenesis of a variety of neurodegenerative diseases. In this study, we examined the expression of C3, the central component of complement, in the brains of transgenic mice with constitutive astrocyte expression of the proinflammatory cytokine, interleukin-6 (IL-6). Immunohistochemistry studies demonstrated elevated deposition of C3 in the brains of transgenic animals compared with normal animals. Northern blot analysis of mRNA from brain and other tissues demonstrated an age-related increase in C3 gene expression only in the brains of transgenic animals, indicative of local synthesis. In situ hybridization studies revealed coincidence between C3 and IL-6 transgene expression, as well as areas of neuronal and white matter damage observed in cerebellum and hind brain. Furthermore, C3 mRNA expression was observed in ependymal cells, perivascular mononuclear cells, endothelial cells, and scattered cells throughout the white matter and the brain stem. The overlap in C3 mRNA expression with areas of pathology suggests that complement may contribute to the inflammation and cellular injury observed in this model. The transgenic mice used in these studies provide a novel and valuable tool for examining the role of complement in central nervous system pathobiology.

Animals↗
PubMed2003

Differential transgene expression in brain cells in vivo and in vitro from AAV-2 vectors with small transcriptional control units.

Adeno-associated- (AAV) based vectors are promising tools for gene therapy applications in several organs, including the brain, but are limited by their small genome size. Two short promoters, the human synapsin 1 gene promoter (hSYN) and the murine cytomegalovirus immediate early promoter (mCMV), were evaluated in bicistronic AAV-2 vectors for their expression profiles in cultured primary brain cells and in the rat brain. Whereas transgene expression from the hSYN promoter was exclusively neuronal, the murine CMV promoter targeted expression mainly to astrocytes in vitro and showed weak transgene expression in vivo in retinal and cortical neurons, but strong expression in thalamic neurons. We propose that neuron specific transgene expression in combination with enhanced transgene capacity will further substantially improve AAV based vector technology.

Animals↗
PubMed2001

Gene expression physiology and pathophysiology of the immune system.

Genomic-scale gene expression profiling can reveal cellular physiology with unprecedented richness. This technology is being used to define the gene expression targets of individual regulatory proteins and signaling pathways. Comprehensive databases of gene expression measurements can be used to understand the pathological mechanisms underlying disease processes.

Animals↗
PubMed2001

Over-expression of DREF in the Drosophila wing imaginal disc induces apoptosis and a notching wing phenotype.

BACKGROUND: DNA replication-related element binding factor (DREF) has been suggested to be involved in regulation of DNA replication- and proliferation-related genes in Drosophila. While the effects on the mutation in the DNA replication-related element (DRE) in cultured cells have been studied extensively, the consequences of elevating wild-type DREF activity in developing tissues have hitherto remained unclear. RESULTS: We over-expressed DREF in the wing imaginal disc using a GAL4-UAS targeted expression system in Drosophila. Over-expression of DREF induced a notching wing phenotype, which was associated with ectopic apoptosis. A half reduction of the reaper, head involution defective and grim gene dose suppressed this DREF-induced notching wing phenotype. Furthermore, this was also the case with co-expression of baculovirus P35, a caspase inhibitor. In addition, over-expression of the 32 kDa boundary element-associated factor (BEAF-32), thought to compete against DREF for common binding sites in genomic regions, rescued the DREF-induced notching wing phenotype, while a half reduction of the genomic region, including the BEAF-32 gene, exerted enhancing effects. To our knowledge, this is the first evidence for a genetic interaction between DREF and BEAF-32. CONCLUSION: The DREF-induced notching wing phenotype is caused by induction of apoptosis in the Drosophila wing imaginal disc.

Animals↗
PubMed2003

NF kappa B controls the balance between Fas and tumor necrosis factor cell death pathways during T cell receptor-induced apoptosis via the expression of its target gene A20.

Activation-induced cell death (AICD), a term originally coined for the anti-CD3-induced apoptosis of T cell hybridomas and thymocytes, is predominantly driven by death receptors and has been involved in the control of autoreactive T cells in the periphery. In the Do-11.10 T cell hybridoma model of AICD, activation of the T cell receptor (TCR) results in Fas-dependent apoptosis. Here, we show that inhibition of the transcription factor nuclear factor kappa B (NF kappa B) in Do-11.10 cells resulted in increased sensitivity to TCR-mediated apoptosis, correlating with defective induction of the anti-apoptotic NF kappa B target gene A20. Stable expression of the zinc finger protein A20 in NF kappa B-negative Do-11.10 cells rescued the phenotype. TCR activation in NF kappa B-deficient Do-11.10 cells resulted predominantly in tumor necrosis factor (TNF) receptor 2 (TNFR2)-dependent bystander cell death rather than classical Fas-dependent AICD. Strikingly, A20 blocked TNF-mediated apoptosis and simultaneously restored TCR-induced Fas-dependent AICD. In addition, NF kappa B downstream of TNFR was required for up-regulation of Fas expression by endogenous TNF secreted in response to TCR stimulation. Together, these results suggest that NF kappa B can play both pro- and anti-apoptotic roles during AICD. We propose that NF kappa B controls the balance between Fas and TNF cell death pathways during AICD via the expression of the zinc finger protein A20.

Animals↗
PubMed2004

Expression microdissection: operator-independent retrieval of cells for molecular profiling.

Tissue microdissection is an important method for the study of disease states. However, it is difficult to perform high-throughput molecular analysis with current techniques. We describe here a prototype version of a novel technique (expression microdissection) that allows for the procurement of desired cells via molecular targeting. Expression microdissection (xMD) offers significant advantages over available methods, including an increase in dissection speed of several orders of magnitude. xMD may become a valuable tool for investigators studying cancer or other disease states in patient specimens and animal models.

Animals↗
PubMed2005

Regulation of human immunodeficiency virus type 1 envelope glycoprotein fusion by a membrane-interactive domain in the gp41 cytoplasmic tail.

Truncation of the human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) gp41 cytoplasmic tail (CT) can modulate the fusogenicity of the envelope glycoprotein (Env) on infected cells and virions. However, the CT domains involved and the underlying mechanism responsible for this "inside-out" regulation of Env function are unknown. HIV and SIV CTs are remarkably long and contain amphipathic alpha-helical domains (LLP1, LLP2, and LLP3) that likely interact with cellular membranes. Using a cell-cell fusion assay and a panel of HIV Envs with stop codons at various positions in the CT, we show that truncations of gp41 proximal to the most N-terminal alpha helix, LLP2, increase fusion efficiency and expose CD4-induced epitopes in the Env ectodomain. These effects were not seen with a truncation distal to this domain and before LLP1. Using a dye transfer assay to quantitate fusion kinetics, we found that these truncations produced a two- to fourfold increase in the rate of fusion. These results were observed for X4-, R5-, and dual-tropic Envs on CXCR4- and CCR5-expressing target cells and could not be explained by differences in Env surface expression. These findings suggest that distal to the membrane-spanning domain, an interaction of the gp41 LLP2 domain with the cell membrane restricts Env fusogenicity during Env processing. As with murine leukemia viruses, where cleavage of a membrane-interactive R peptide at the C terminus is required for Env to become fusogenic, this restriction of Env function may serve to protect virus-producing cells from the membrane-disruptive effects of the Env ectodomain.

Amino Acid Sequence↗
PubMed2003

Fusion protein system designed to provide color to aid in the expression and purification of proteins in Escherichia coli.

We have designed and constructed a new fusion expression vector (pKW32), which contains the His-tagged Vitreoscilla hemoglobin (VHb) coding gene upstream of the multiple cloning site. The pKW32 vector was designed to express target proteins as VHb fusions, which can be purified in one step by affinity chromatography. Due to the color of the heme in VHb, the VHb-fused target proteins have a red color that provides a visual aid for estimating their expression level and solubility. The red color can also be used as a visual marker throughout purification, while the concentration of the fusion protein can be determined by measuring the amount of VHb using carbon monoxide difference spectra. In addition, because of inherently high solubility of VHb, the fusion can increase the solubility of sparingly soluble target proteins. Target proteins can be easily separated from His-tagged VHb due to the presence of a thrombin-cleavage site between them. A mutant VHb, the soluble domain of Vitreoscilla cytochrome bo subunit II, and HIV integrase expressed and purified using the pKW32 system have native function. In addition, the integrase, which is known to be difficult to purify because of low solubility, was purified simply and without solubilizing agents using our system.

Base Sequence↗
PubMed2006

Lens cell targetting for gene therapy of prevention of posterior capsule opacification.

Posterior capsule opacification is the main complication of cataract surgery. Using adenovirus-mediated gene transfer, we recently reported that it was feasible to prevent PCO by overexpressing pro-apoptotic molecules such as pro-caspase 3 or Bax in the residual lens epithelial cells post-cataract surgery. However, this approach is feasible only if gene transfer can be restricted to the residual cells responsible for PCO. Initially, we tested an adenovirus (human serotype 5, HAd5), a lentivirus (HIV) and an oncoretrovirus (MLV) vector for the their in vivo transduction efficiency of rabbit lens cells. We found that HAd5 vectors were the most efficient (>90% of the cells could be transduced). Six potential lens-specific promoters were then cloned into HAd5 vectors and assayed for their ability to target expression to a specific population of cells, using in vitro, ex vivo and in vivo rabbit tissues and human lens capsular bags. We found that the LEP503, MIP and Filensin promoters induced strong lens-specific expression of a reporter gene, in human lens cells. Following this ex vivo assay, we showed in a rabbit PCO model that gene transfer could be spatially restricted to the capsular bag by confining the vector with Matrigel. Our combined approach using a lens-specific promoter and a biocompatible gel should render feasible a novel therapeutic strategy for PCO that targets the remaining lens cells.

Adenoviridae↗
PubMed1996

Identification and characterization of the genes of Enterococcus faecalis plasmid pCF10 involved in replication and in negative control of pheromone-inducible conjugation.

The prgB gene of the Enterococcus faecalis pheromone-inducible conjugative plasmid pCF10 encodes the surface protein Asc10. This protein mediates cell aggregation and its expression results in high-frequency transfer of the plasmid from donor to recipient. To identify the minimum region necessary for negative regulation of prgB expression, target plasmids were constructed containing a recently identified positive control region and a prgB::lacZ transcriptional fusion; expression of prgB in cells carrying these plasmids was thus verified by beta-galactosidase assay. The target plasmids were used in genetic studies with compatible plasmids containing cloned pCF10 genes supplying putative negative control functions to define the minimum region of pCF10 required for shutdown of prgB expression in the absence of exogenous pheromone. The minimum segment required for negative control, as indentified by deletion analysis, was a 6.9-kb region extending from the 5' end of a gene called prgN, through a previously identified gene, prgX. The DNA in this region, which had not been previously characterized (2.85 kb), was sequenced, and several potential regulatory genes and plasmid replication genes were identified. Genetic analysis indicated that the prgN, -Y, and -X genes are involved in negative control; prgW may also play a role in negative control, since it appeared to be required for expression of prgY. prgX, or a closely adjacent DNA sequence, acted in cis. The region of pCF10 containing negative control genes was also shown to function as an autonomous replicon in E. faecalis.

Amino Acid Sequence↗
PubMed2005

Heterotypic interaction of CRTAM with Necl2 induces cell adhesion on activated NK cells and CD8+ T cells.

NK cells and CD8+ T cells exhibit cytotoxicity and cytokine production upon recognizing target cells through cell-cell interaction. We screened the molecules involved in the recognition and regulation of these cells using cDNA subtraction between naive and activated NK cells. We identified class I-restricted T cell-associated molecule (CRTAM), a two Ig domain-bearing surface receptor, as a molecule rapidly and transiently expressed on NK cells and CD8+ T cells upon activation. CRTAM is expressed as a dimer on the cell surface, and its expression is transcriptionally regulated. Using an expression-cloning system, we then further identified Nectin-like (Necl) molecule 2, a three Ig domain-containing receptor, as a ligand of CRTAM. While Necl2 mediates homotypic interaction, CRTAM interacts with Necl2 but not with CRTAM itself. The heterotypic CRTAM-Necl2 interaction has a higher affinity than the homotypic Necl2 interaction. Although there was no clear alteration in the cytotoxic function of the NK cells and CD8+ T cells against the Necl2-expressing target cells, T cells expressing CRTAM tightly bound to Necl2-expressing cells. CRTAM+ cells did not induce homotypic aggregation but they did exert strong heterotypic binding with Necl2+ cells, which was inhibited by the addition of the CRTAM-Ig fusion protein. These results suggest that the heterotypic interaction between CRTAM and Necl2 plays an important role in the adhesion, interaction or migration of NK cells and CD8+ T cells upon stimulation.

Animals↗
PubMedDate not supplied

Transgenic targeting of neuroendocrine peptide genes in the hypothalamic-pituitary axis.

A large number of neuroendocrine peptide genes have been tested for their ability to target expression to the hypothalamus and pituitary in transgenic mice. This has resulted in a number of powerful applications, for example, ablation or immortalization of specific cell types, and analysis of transcription regulatory sequences. The greatest amount of success in targeting cells of the neuroendocrine axis has been in the pituitary and has utilized regulatory sequences of genes that are normally expressed in pituitary. Greater difficulties have been encountered in directing expression to specific neurons in the hypothalamus. A primary goal of this review is to consider collectively the data obtained by a number of laboratories in order to draw conclusions about the general sequence requirements for achieving cell-specific expression. The data suggest that the mechanisms controlling cell-specific expression of neuropeptide genes in the hypothalamus are complex and involve multiple regulatory elements that may reside within the gene or many kilobases away from the promoter. These elements act positively and negatively in different cells to enhance or restrict expression, and may include sequences that shield a transgene from regulatory influences of other genes near the point of chromosomal insertion.

Animals↗
PubMed1999

Cloning and analysis of small cytoplasmic leucine-rich repeat protein (SCLP), a novel, phylogenetically-conserved protein that is dramatically up-regulated during the programmed death of moth skeletal muscle.

We used the abdominal intersegmental muscles (ISMs) of the moth Manduca sexta as a source of transcripts that are dramatically up-regulated during programmed cell death. One of these transcripts, Small Cytoplasmic Leucine-Rich Repeat Protein (SCLP), encodes a protein of approximately 24 kD that contains four perfect and two imperfect leucine-rich repeat (LRR) motifs. DNA sequence database analysis suggests that SCLP is a phylogenetically-conserved gene of unknown function. Both Northern and Western blots demonstrated that SCLP is expressed in the ISMs at all stages examined, but increases greater than 10-fold when the cells become committed to die. This increase in expression is regulated by the same change in the circulating ecdysteroid titer that controls death. Low levels of SCLP expression are also seen in flight muscle and fat body, but not in ovary, male sexual accessory gland, or Malpighian tubules. Immunohistochemical analysis demonstrates that SCLP is a cytoplasmic protein. Western blot analysis of proteins from the fly Drosophila suggests that an SCLP-related protein is expressed at the larval and pupal stages, but not in embryos or adults. Targeted expression of moth SCLP to a variety of different tissues in Drosophila using the Gal4/UAS P element system failed to generate an overt phenotype. These data are interpreted as suggesting that whereas SCLP presumably plays an important role in programmed cell death of muscle, perhaps by acting as an adaptor protein, its expression is insufficient to initiate death by itself.

Amino Acid Sequence↗
PubMed2006

Identification of alkaline proteins that are differentially expressed in an overgrowth-mediated growth arrest and cell death of Escherichia coli by proteomic methodologies.

The available Escherichia coli genome sequences offer an opportunity to further expand our understanding of this bacterium. In the current study, we present a rapid method for the isolation of bacterial alkaline proteins using acid incubation, purification and protein array by 2-DE, followed by protein identification using MS. Fifty-seven proteins were randomly chosen, in which 55 were identified by a database searching of MS data. The searching results showed that most of these alkaline proteins were involved in special functions within the cell, suggesting that alkaline proteome is an ideal fraction for an understanding of their special functions. Furthermore, alkaline proteomes were compared between the period of majority live bacteria (18-h culture), the period of similar amount of live and dead bacteria (30-h culture) and the period of majority dead bacteria (48-h culture). Six proteins were identified as differentially expressed targets, in which putative transcriptional regulator and superoxide dismutase genes were cloned and expressed for antiserum preparations. The antisera were applied for the confirmation of results obtained from 2-DE. The presented data clearly reveal that alkaline proteome analysis by 2-DE with MS plays an important role in the understanding of protein functions within the cell, and six alkaline proteins are determined as key ones in an overgrowth-mediated growth cycle of E. coli.

Cell Death↗