Infancy onset hereditary spastic paraplegia associated with a novel atlastin mutation.
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Adolescent↗
Biomedical subjects
A Daga
Publications and source records attributed to A Daga.
Enhanced engraftment of EPO-transduced human bone marrow stromal cells transplanted in a 3D matrix in non-conditioned NOD/SCID mice.
Intravenous infusion of bone marrow stromal cells (BMSCs) has been proposed as a means to support hematopoiesis in bone marrow transplantation or as a vehicle for gene therapy. However, it seems that this route of injection leads to engraftment of a small proportion of BMSCs, possibly because they are unable to cross the endothelial barrier. We have transplanted human BMSCs, ex vivo expanded and transduced with a retrovirus encoding the human erythropoietin gene, either intravenously or subcutaneously with or without a tridimensional scaffold in non-conditioned NOD/SCID mice. Efficiency of engraftment was evaluated monitoring the hematocrit levels. Systemic infusion never significantly increased hematocrit levels, whereas subcutaneous transplantation of the same number of cells induced an important increase of the hematocrit (approximately 70%) for at least 2 months. A substantial increase in the length of the response was observed when cells were subcutaneously transplanted in a tridimensional scaffold. To determine whether the transient effect was due to cell loss or to reduction in expression, the cells implanted into a tridimensional scaffold were recovered, expanded in vitro, and re-implanted in a new group of mice. Again the hematocrit levels rose 2 weeks after transplantation ( approximately 70%). These results demonstrate that ex vivo expanded human BMSCs are not quantitatively transplantable by systemic infusion in non-conditioned recipients, whereas the local implantation into a tridimensional scaffold allows long-term engraftment and efficient expression of a foreign gene.
Adipocytes↗
DNase I mediates internucleosomal DNA degradation in human cells undergoing drug-induced apoptosis.
Internucleosomal DNA fragmentation following the activation of endonucleases is the common end point of apoptosis. DNase I, a Ca(2+) / Mg(2+)-dependent endonuclease ubiquitously expressed in mammalian tissues, is believed to play a role in this process. To analyze the in vivo function of this enzyme in human cells, we have generated a cell line with targeted disruption of the DNase I gene, as well as several stable cell lines which overexpress the DNase I gene. Inactivation of the human DNase I gene was obtained in the Jurkat T cell clone JA3, characterized by high susceptibility to apoptotic cell death induced by pharmacological stimuli. JA3 cells, after disruption of the DNase I gene, became resistant to apoptotic stimuli. DNase I was overexpressed in the human cell lines JA3, K562 (erythroleukemia), M 14 (melanoma) and CEM (T cell lymphoma). Remarkably, stable overexpression of DNase I gene resulted in accelerated apoptosis in JA3 cells and induced apoptosis in K562, CEM and M14 cell lines, which are otherwise resistant to internucleosomal DNA degradation following pharmacological stimuli. Our study provides the first in vivo evidence that DNase I mediates internucleosomal DNA degradation in human cells undergoing drug-induced apoptosis.
Apoptosis↗
Emx2 in adult neural precursor cells.
Emx2 is a vertebrate homeobox gene involved in the control of the central nervous system development. In the formation of cerebral cortex, Emx2 expression is restricted mainly to the germinal ventricular zone fading away in the first postmitotic neurons. This expression pattern, the severe impairment of cortex organization and the size in mutant mice suggest a role of Emx2 in the control of proliferation and migration of neural precursor cells. The observed persistence of Emx2 expression in adult neurogenic areas in vivo is here confirmed at later stages. We also find that Emx2 is expressed at high levels in adult neural stem cells (ANSCs) in vitro and is down modulated upon differentiation. Overexpression of Emx2 gene in ANSCs has an anti-proliferative effect but it does not influence a particular differentiation pathway. Our results suggest that Emx2 may act promoting an asymmetric mode of cell division thereby increasing the size of a transit amplifying population.
Animals↗
The retroviral transduction of HOXC4 into human CD34(+) cells induces an in vitro expansion of clonogenic and early progenitors.
OBJECTIVE: +HOX genes are expressed in the hematopoietic system and increasing data point to their involvement in the control of proliferation and/or differentiation. Genes belonging to the C cluster are preferentially expressed in developing and differentiated lymphoid lineages. However, recent studies demonstrated, by RT-PCR, that the HOXC4 gene is also actively transcribed in the most undifferentiated hematopoietic cells (CD34(+)38(low)) and in more mature myeloid and erythroid progenitors. We evaluated the expression of HOXC4 protein on human CD34(+) cells and the in vitro effect of its overexpression on proliferation and differentiation. MATERIALS AND METHODS: We assessed the expression of HOXC4 on human CD34(+) cells using a polyclonal antibody raised against the C-terminal portion of the protein expressed using the baculovirus system. Overexpression of HOXC4 in human CD34(+) cells was obtained by retroviral gene transfer; its effect on clonogenic (CFU-GM, BFU-E, and CFU-GEMM) and early progenitors (LTC-IC) was evaluated. RESULTS: The HOXC4 protein is indeed expressed in human CD34(+) cells, and its overexpression in human CD34(+) cells increases the proliferation potential of clonogenic and early progenitors. CFU-GM showed a median threefold expansion (range: 1.1-19.4; p < 0.002) compared with control transduced with the vector alone. The increment of BFU-E was higher (median ninefold, range 2.5-35; p < 0. 0009) and erythroid colonies presented a larger size with normal morphology. An even more marked effect was observed on LTC-IC (median 13, onefold; range 4.1-102.1, p < 0.0001). CONCLUSION: We demonstrate that HOXC4 is expressed in CD34(+) cells and that its overexpression induces an in vitro expansion of committed as well as very early hematopoietic progenitors. The most striking effect was obtained on LTC-IC with an expansion of 13.1-fold. The enforced expression of HOXC4 induced a significant increase (p < 0.009) in the number of erythroid colonies compared with CFU-GM, although without perturbing, at least in vitro, the maturation program of the cells. On the other hand, the effect of the gene overexpression did not induce any skewing in the colony types derived from the myeloid lineage.
Antigens, CD34↗
Extracellular cyclic ADP-ribose increases intracellular free calcium concentration and stimulates proliferation of human hemopoietic progenitors.
Cyclic ADP-ribose (cADPR) is a universal second messenger that regulates many calcium-related cellular events by releasing calcium from intracellular stores. Since these events include enhanced cell proliferation and since the bone marrow harbors both ectoenzymes that generate cADPR from NAD(+) (CD38 and BST-1), we investigated the effects of extracellular cADPR on human hemopoietic progenitors (HP). Exposure of HP to 100 microM cADPR for 24 h induced a significant increase in colony output (P<0.01) and colony size (P<0.003). A horizontal expansion of HP, as demonstrated by a markedly increased replating efficiency in semisolid medium (up to 700 times compared to controls), was also observed, indicating that cADPR priming can affect cell growth for multiple generations over several weeks after exposure. Influx of extracellular cADPR into the cells was demonstrated, and a causal relationship between the functional effects and the increase of intracellular free calcium concentration induced by cADPR on HP was established through the use of specific antagonists. Similar effects on HP were produced by nanomolar concentrations of the nonhydrolyzable cADPR analog 3-deaza-cADPR. These data demonstrate that extracellular cADPR behaves as a cytokine enhancing the proliferation of human HP, a finding that may have biomedical applications for the ex vivo expansion of hemopoietic cells.
Adenosine Diphosphate Ribose↗
Expression of CD38 increases intracellular calcium concentration and reduces doubling time in HeLa and 3T3 cells.
CD38 is a bifunctional ectoenzyme, predominantly expressed on hematopoietic cells during differentiation, that catalyzes the synthesis (cyclase) and the degradation (hydrolase) of cyclic ADP-ribose (cADPR), a powerful calcium mobilizer from intracellular stores. Due to the well established role of calcium levels in the regulation of apoptosis, proliferation, and differentiation, the CD38/cADPR system seems to be a likely candidate involved in the control of these fundamental processes. The ectocellular localization of the cyclase activity, however, contrasts with the intracellular site of action of cADPR. Here we demonstrate that ectocellular expression of human CD38 in CD38(-) HeLa and 3T3 cells results in intracellular CD38 substrate (NAD+ + NADH) consumption and product (cADPR) accumulation. Furthermore, a causal relationship is established between presence of intracellular cADPR, partial depletion of thapsigargin-sensitive calcium stores, increase in basal free cytoplasmic calcium concentration, and decrease of cell doubling time. The significant shortening of the S phase in CD38(+) HeLa cells, as compared with controls, demonstrates an effect of intracellular cADPR on the mammalian cell cycle.
3T3 Cells↗
Lozenge is expressed in pluripotent precursor cells and patterns multiple cell types in the Drosophila eye through the control of cell-specific transcription factors.
In the developing Drosophila eye, individual cell fates are specified when general signaling mechanisms are interpreted in the context of cell-specific transcription factors. Lozenge, a Runt/AML1/CBFA1-like transcription factor, determines the fates of a number of neuronal and non-neuronal cells by regulating the expression of multiple fate-determining transcription factors. The Lozenge protein is expressed in the nuclei of the cells that it patterns and also in their undifferentiated precursors. An enhancer element located within the second intron of the lozenge gene is responsible for its eye-specific expression. Lozenge is not itself a cell-specific transcription factor, rather it prepatterns the eye disc by positioning cell-specific factors in their appropriate locations.
Animals↗
Interactions of Drosophila Cbl with epidermal growth factor receptors and role of Cbl in R7 photoreceptor cell development.
The human proto-oncogene product c-Cbl and a similar protein in Caenorhabditis elegans (Sli-1) contain a proline-rich COOH-terminal region that binds Src homology 3 (SH3) domains of proteins such as the adapter Grb2. Cb1-Grb2 complexes can be recruited to tyrosine-phosphorylated epidermal growth factor (EGF) receptors through the SH2 domain of Grb2. Here we identify by molecular cloning a Drosophila cDNA encoding a protein (Drosophila Cbl [D-Cbl]) that shows high sequence similarity to the N-terminal region of human c-Cbl but lacks proline-rich sequences and fails to bind Grb2. Nonetheless, in COS-1 cells, expression of hemagglutinin epitope-tagged D-Cbl results in its coimmunoprecipitation with EGF receptors in response to EGF. EGF also caused tyrosine phosphorylation of D-Cbl in such cells, but no association of phosphatidylinositol 3-kinase was detected in assays using anti-p85 antibody. A point mutation in D-Cbl (G305E) that suppresses the negative regulation of LET-23 by the Cbl homolog Sli-1 in C. elegans prevented tyrosine phosphorylation of D-Cbl as well as binding to the liganded EGF receptor in COS-1 cells. Colocalization of EGF receptors with both endogenous c-Cbl or expressed D-Cbl in endosomes of EGF-treated COS-1 cells is also demonstrated by immunofluorescence microscopy. In lysates of adult transgenic Drosophila melanogaster, GST-DCbl binds to the tyrosine-phosphorylated 150-kDa torso-DER chimeric receptor. Expression of D-Cbl directed by the sevenless enhancer in intact Drosophila compromises severely the development of the R7 photoreceptor neuron. These data suggest that despite the lack of Grb2 binding sites, D-Cbl functions as a negative regulator of receptor tyrosine kinase signaling in the Drosophila eye by a mechanism that involves its association with EGF receptors or other tyrosine kinases.
Adaptor Proteins, Signal Transducing↗
Patterning of cells in the Drosophila eye by Lozenge, which shares homologous domains with AML1.
The lozenge (lz) gene encodes a transcription factor involved in prepatterning photoreceptor precursors in the developing Drosophila eye. The central region of the predicted Lz protein product is homologous to AML1, a transcription factor associated with human leukemias, and to the Drosophila protein Runt. We show here that Lz plays a crucial role in governing the fate of two groups of cells that are born in a single round of mitosis in the larval eye disc. Lz helps define a subset of these cells as an equipotential group that is competent to respond to the Sevenless developmental signal. This is achieved by negative regulation of seven-up, a member of the steroid hormone receptor superfamily in these cells. In contrast, in a second group of cells, the Lz protein confers proper photoreceptor identity by positively regulating the homeo box gene Bar. Additionally, our genetic analysis suggests that Lz interacts with the Ras pathway to determine photoreceptor cell fate. This study suggests that the strategies involved in cell fate determination in the Drosophila eye are remarkably similar to those utilized during vertebrate hematopoietic development and require the coordinate action of growth factor and AML1-like pathways.
Alleles↗
Expression of runtB is modulated during chondrocyte differentiation.
The runt locus in Drosophila encodes a nuclear protein involved in embryo segmentation, sex determination/X dosage compensation, and neurogenesis. runt homologues have been identified in higher vertebrates. The encoded proteins share a domain of 128 amino acids called the runt domain. It has been reported that this domain mediates DNA binding and heterodimerization. Here, we analyze runtB expression during chondrocyte differentiation "in vitro" and "in vivo." We have first isolated, from a chondrocyte library, a cDNA clone coding for a runtB chicken homologue and containing a complete open reading frame. The predicted protein product is 84% identical to the mouse PEBP2alphaB2 isoform. By RT-PCR analysis we have also cloned the chicken cDNA fragment coding for delta alphaB2, the exon sequence included in the B1 isoform mRNA. On Northern blot analysis of cultured chondrocytes, runtB mRNA levels increase dramatically with the transition from stage 0 (dedifferentiated) to stages I and II (hypertrophic chondrocytes). Moreover, runt polypeptides were demonstrated in chondrocytes both in vivo and in vitro. These results suggest that runt plays a role in chondrogenic differentiation.
Amino Acid Sequence↗
Spontaneously produced anti-DNA/DNase I autoantibodies modulate nuclear apoptosis in living cells.
Apoptosis has received increased attention over the past decade, and it is established as an essential process in physiological and disease states. Much effort has been devoted to understanding the intracellular mechanisms culminating in apoptosis; intense investigation has also focused on its role during inflammation. Despite these efforts, these events remain incompletely understood. It has been suggested that the Ca(2+)- and Mg2+-dependent endonuclease that mediates DNA fragmentation is DNase I; however, the precise role of DNase I during apoptosis has been debated. Recent observations using anti-DNA antibodies derived from autoimmune mice (MRL-lpr/lpr) provided both the means and the reagents to approach these issues in a more direct manner. We previously discovered that many anti-DNA antibodies cross-react with DNase I, and a subset of these Ig inhibited DNase I enzymatic activity in vitro. Serendipitously, in separate studies, a subset of these antibodies were observed to enter and localize within the nuclei of living cells. The aim of the present investigation was to determine whether these nuclear-localizing anti-DNA antibodies could interact with DNase I in living cells. We found that, once internalized, these autoantibodies bound DNase I and inhibited activity of the enzyme. Furthermore, living cells containing the intracellular antibodies appeared resistant to apoptotic stimuli; both morphological features of nuclear apoptosis and DNA fragmentation were inhibited. These results support a pivotal role for DNase I in apoptosis, and they provide a novel paradigm for autoantibody-mediated inflammatory disease.
Antibodies, Antinuclear↗
In vivo functional analysis of the Ras exchange factor son of sevenless.
The Son of sevenless (Sos) protein functions as a guanine nucleotide transfer factor for Ras and interacts with the receptor tyrosine kinase Sevenless through the protein Drk, a homolog of mammalian Grb2. In vivo structure-function analysis revealed that the amino terminus of Sos was essential for its function in flies. A molecule lacking the amino terminus was a potent dominant negative. In contrast, a Sos fragment lacking the Drk binding sites was functional and its activity was dependent on the presence of the Sevenless receptor. Furthermore, membrane localization of Sos was independent of Drk. A possible role for Drk as an activator of Sos is discussed and a Drk-independent interaction between Sos and Sevenless is proposed that is likely mediated by the pleckstrin homology domain within the amino terminus.
Animals↗
Expression of runt in the mouse embryo.
The Drosophila runt locus controls early events in embryogenesis. A human homologue (CBFA2) was originally identified because of its involvement in the t(8;21) associated with a subtype of acute myeloid leukaemia. The phylogenetically conserved region (runt box) was reported to correspond to a DNA binding domain. In order to investigate whether runt also plays a role in mammalian development, we have conducted a preliminary survey of its expression in the mouse embryo. Expression in embryonic tissues was detected starting from day 9.2 post coitum. From day 10.5 post coitum, highest levels are found in the neural tube, sensory ganglia, specialised sensory epithelial structures (olfactory and gustatory mucosa, follicles of the vibrissae), all chondrogenic centres (both of neural crest and of mesodermal origin), and the genital system (the gonad, the paramesonephros, and the genital tubercle). Unambiguous expression in the haemopoietic system could be established for the thymus. The data suggest a pleiotropic role for mammalian runt in embryogenesis.
Animals↗
Resolving the sevenless pathway using sensitized genetic backgrounds.
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Animals↗
Molecular characterization of the transcription termination factor from human mitochondria.
The transcription termination factor (mTERF), which plays a central role in the control of mitochondrial rRNA and mRNA synthesis in mammalian mitochondria, has been previously identified and purified by DNA affinity chromatography from a human mitochondrial lysate (Kruse, B., Narasimhan, N., and Attardi, G. (1989) Cell 58, 391-397). In the present work, this factor has been characterized as to its protein composition and the activities of the protein components. Three polypeptides, two of approximately 34-kDa molecular mass and one of approximately 31 kDa, were shown to be associated with the specific DNA binding and footprinting activity of the factor, with the 31-kDa component having a much lower affinity for the recognition sequence than the 34-kDa components. On the other hand, the transcription termination activity, as assayed in an in vitro system, was found to be associated exclusively with the two 34-kDa polypeptides. Mass spectroscopic analysis of tryptic peptides derived from highly purified polypeptides indicated that all three polypeptides share regions with common sequences. The evidence obtained suggests that differential phosphorylation is not responsible for the difference in electrophoretic mobility of the three polypeptides.
Base Sequence↗
Translocation breakpoints are clustered on both chromosome 8 and chromosome 21 in the t(8;21) of acute myeloid leukemia.
The t(8;21)(q22;q22) is consistently associated with acute myeloid leukemia (AML) M2. Recent data have suggested that breakpoints on chromosome 21 are clustered within a single intron of a novel gene, AML1, just downstream of a region of homology to the runt gene of D melanogaster. In this report, we confirm rearrangement at the same location in at least 12 of 18 patients with t(8;21). Furthermore, we have isolated recombinant clones spanning the breakpoint regions on both the der(8) and the der(21) from one patient. By using a chromosome 8 probe derived from these clones, we show that t(8;21) breakpoints are also clustered on chromosome 8.
Acute Disease↗
Molecular analysis of the gamma delta T-cell receptor repertoire in normal human skin and in Oriental cutaneous leishmaniasis.
The extent of diversity of the gamma delta T-cell receptor (TCR) in normal human skin and Oriental Cutaneous Leishmaniasis (OCL) was examined by molecular analysis of the variable (V) delta gene segment, junctional (J) delta gene segment and junctional regions. To examine the expression of TCR delta genes, segments of gamma delta T lymphocytes, DNA isolated from normal human skin and from OCL were subjected to enzymatic gene amplification by the polymerase chain reaction (PCR) method using TCR V delta- and J delta-specific oligonucleotides as primers. PCR amplification using these primers indicated that the V delta 2 gene segment was predominantly used by gamma delta T lymphocytes in both normal human skin and OCL. To determine the extent of junctional diversity in the delta gene of gamma delta T cells in normal human skin and OCL, we sequenced the nucleic acid sequences corresponding to the V delta 2/J delta 1 junctional regions. Sequence analysis of junctional regions demonstrated broad junctional diversity in normal skin but only limited diversity in OCL. Our findings support the hypothesis that skin gamma delta T lymphocytes may derive from a fetal subset of gamma delta T lymphocytes that leaves the thymus early and colonizes the periphery. The limited junctional diversity demonstrated in OCL lesions indicates that gamma delta T cells can undergo oligoclonal expansion following recognition of a specific ligand and supports the idea that junctional regions are important in the recognition of antigenic determinant.
Amino Acid Sequence↗