Biomedical subjects

G Caruana

Publications and source records attributed to G Caruana.

9 recordsLinked to original sources

PubMed2005

Temporal and spatial transcriptional programs in murine kidney development.

We have performed a systematic temporal and spatial expression profiling of the developing mouse kidney using Compugen long-oligonucleotide microarrays. The activity of 18,000 genes was monitored at 24-h intervals from 10.5-day-postcoitum (dpc) metanephric mesenchyme (MM) through to neonatal kidney, and a cohort of 3,600 dynamically expressed genes was identified. Early metanephric development was further surveyed by directly comparing RNA from 10.5 vs. 11.5 vs. 13.5dpc kidneys. These data showed high concordance with the previously published dynamic profile of rat kidney development (Stuart RO, Bush KT, and Nigam SK. Proc Natl Acad Sci USA 98: 5649-5654, 2001) and our own temporal data. Cluster analyses were used to identify gene ontological terms, functional annotations, and pathways associated with temporal expression profiles. Genetic network analysis was also used to identify biological networks that have maximal transcriptional activity during early metanephric development, highlighting the involvement of proliferation and differentiation. Differential gene expression was validated using whole mount and section in situ hybridization of staged embryonic kidneys. Two spatial profiling experiments were also undertaken. MM (10.5dpc) was compared with adjacent intermediate mesenchyme to further define metanephric commitment. To define the genes involved in branching and in the induction of nephrogenesis, expression profiling was performed on ureteric bud (GFP+) FACS sorted from HoxB7-GFP transgenic mice at 15.5dpc vs. the GFP- mesenchymal derivatives. Comparisons between temporal and spatial data enhanced the ability to predict function for genes and networks. This study provides the most comprehensive temporal and spatial survey of kidney development to date, and the compilation of these transcriptional surveys provides important insights into metanephric development that can now be functionally tested.

Animals↗

PubMed2003

Changes in serum electrolytes during treatment of patients in liver failure with molecular adsorbent recirculating system.

PURPOSE: To study the effect of MARS on serum electrolytes during liver failure. DESIGN: Twenty-three patients admitted to a quaternary health care facility from September 2000 to May 2002, 22 adults and 1 child, 11 males (48%) and 12 females (52%), age 15-70 (median 53), treated with MARS for: 12 acute-on-chronic liver failure (52%); 4 fulminant hepatic failure (17%); 3 intractable pruritus (13%); 2 primary-non-function (9%); 2 following major liver resection (9%). PROCEDURES: Sodium, potassium, chloride, phosphorus, calcium, and magnesium were measured in the serum, ultrafiltrate and albumin circuit before and after MARS. STATISTICAL METHODS: A comparison of electrolyte concentrations, before and after MARS, was performed using a paired t test. MAIN FINDINGS: Serum electrolyte concentrations before and after MARS, while statistically significant in some cases, were very small, and of no clinical relevance. CONCLUSION: MARS exchanges potassium, chloride, calcium, and magnesium by ultrafiltration; sodium by the albumin dialysis.

Adolescent↗

PubMed2001

Craniofacial dysmorphogenesis including cleft palate in mice with an insertional mutation in the discs large gene.

The discs large (Dlg) protein, or synapse-associated protein 97 (SAP97), is a member of the membrane-associated guanylate kinase family of multidomain scaffolding proteins which recruits transmembrane and signaling molecules to localized plasma membrane sites. Murine dlg is the homologue of the Drosophila dlg tumor suppressor gene. The loss of dlg function in Drosophila disrupts cellular growth control, apicobasal polarity, and cell adhesion of imaginal disc epithelial cells, resulting in embryonic lethality. In this study, we isolated a mutational insertion in the murine dlg locus by gene trapping in totipotent embryonic stem cells. This insertion results in a truncated protein product that contains the N-terminal three PSD-95/DLG/ZO-1 domains of Dlg fused to the LacZ reporter and subsequently lacks the src homology 3 (SH3), protein 4.1 binding, and guanylate kinase (GUK)-like domains. The Dlg-LacZ fusion protein is expressed in epithelial, mesenchymal, neuronal, endothelial, and hematopoietic cells during embryogenesis. Mice homozygous for the dlg mutation exhibit growth retardation in utero, have hypoplasia of the premaxilla and mandible, have a cleft secondary palate, and die perinatally. Consistent with this phenotype, Dlg-LacZ is expressed in mesenchymal and epithelial cells throughout palatal development. Our genetic and phenotypic analysis of dlg mutant mice suggests that protein-protein interactions involving the SH3, protein 4.1 binding, and/or GUK-like domains are essential to the normal function of murine Dlg within craniofacial and palatal morphogenesis.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Gene trapping of two novel genes, Hzf and Hhl, expressed in hematopoietic cells.

Using an expression gene trapping strategy, we have identified and characterized two novel hematopoietic genes, Hzf and Hhl. Embryonic stem (ES) cells containing a gene trap vector insertion were cultured on OP9 stromal cells to induce hematopoietic differentiation and screened for lacZ reporter gene expression. Two ES clones displaying lacZ expression within hematopoietic cells in vitro were used to generate mice containing the gene trap integrations. Paralleling this in vitro expression pattern, both Hzf and Hhl were expressed in a tissue-specific manner during hematopoietic development in vivo. Hzf encodes a novel protein containing three C(2)H(2)-type zinc fingers predominantly expressed in megakaryocytes and CFU-GEMM. Hhl encodes a novel protein containing a putative phosphotyrosine binding (PTB) domain expressed in megakaryocytes, CFU-GEMM and BFU-E. These results demonstrate the utility of expression trapping to identify novel hematopoietic genes. Future studies of Hzf and Hhl should provide valuable information on the role these genes play during megakaryocytopoiesis.

Amino Acid Sequence↗

PubMed2000

Mastocytosis cells bearing a c-kit activating point mutation are characterized by hypersensitivity to stem cell factor and increased apoptosis.

Mastocytosis is characterized by abnormal infiltration of mast cells into various organs. An activating mutation in c-kit, involving an A --> T substitution at nucleotide 2648 has recently been described in some patients with mastocytosis. We describe a 12-year-old girl with this mutation in her bone marrow cells at diagnosis with a myelodysplastic syndrome (MDS) without evidence of mastocytosis, and then in peripheral blood mononuclear cells 1 year later after the emergence of mastocytosis. The role of the c-Kit receptor and its ligand stem cell factor (SCF) in the pathogenesis of the disease was analysed in marrow cell clonogenic assays. We show that the genetic abnormalities in the patient resulted in factor-independent growth and hypersensitivity of primitive progenitors to SCF, with increased production of mast cells. Increased apoptosis and cluster formation, consistent with the myelodysplastic nature of the disorder, accompanied accumulation of abnormal cells with increasing concentrations of SCF.

Apoptosis↗

PubMed1999

Isoforms of c-KIT differ in activation of signalling pathways and transformation of NIH3T3 fibroblasts.

Alternate splicing of mRNA encoding c-KIT results in isoforms which differ in the presence or absence of four amino acids (GNNK) in the juxtamembrane region of the extracellular domain of the receptor. In this study we show that these isoforms of human c-KIT, expressed at similar levels in NIH3T3 cells, display differential effects on various attributes of transformation. The GNNK- isoform strongly promoted anchorage independent growth (colony formation in semi-solid medium), loss of contact inhibition (focus formation), and led to tumorigenicity in nude mice. In contrast, the GNNK+ isoform elicited colony formation but relatively poor focus formation and no tumorigenicity. Saturation binding analysis indicated that the isoforms do not differ significantly in their affinity for the KIT ligand, Steel Factor (SLF). Negligible ligand-independent receptor phosphorylation was observed in either case but, after ligand stimulation, the GNNK- isoform displayed more rapid and extensive tyrosine autophosphorylation and faster internalization. Both isoforms recruited the p85 subunit of phosphatidylinositol 3-kinase and led to similar phosphorylation of its downstream effector c-Akt, but the GNNK- isoform gave rise to more MAP kinase phosphorylation. Thus the c-KIT isoforms display different signalling characteristics and have different transforming activity in NIH3T3 cells.

3T3 Cells↗

PubMed1998

Expression trapping: identification of novel genes expressed in hematopoietic and endothelial lineages by gene trapping in ES cells.

We have developed a large-scale, expression-based gene trap strategy to perform genome-wide functional analysis of the murine hematopoietic and vascular systems. Using two different gene trap vectors, we have isolated embryonic stem (ES) cell clones containing lacZ reporter gene insertions in genes expressed in blood island and vascular cells, muscle, stromal cells, and unknown cell types. Of 79 clones demonstrating specific expression patterns, 49% and 16% were preferentially expressed in blood islands and/or the vasculature, respectively. The majority of ES clones that expressed lacZ in blood islands also expressed lacZ upon differentiation into hematopoietic cells on OP9 stromal layers. Importantly, the in vivo expression of the lacZ fusion products accurately recapitulated the observed in vitro expression patterns. Expression and sequence analysis of representative clones suggest that this approach will be useful for identifying and mutating novel genes expressed in the developing hematopoietic and vascular systems.

Animals↗

PubMed1998

Transformation of NIH3T3 fibroblasts by the c-Kit receptor tyrosine kinase: effect of receptor density and ligand-requirement.

Ectopic expression of the normal murine receptor tyrosine kinase, c-Kit, in NIH3T3 cells induced many phenotypic changes characteristic of transformation including anchorage-independent growth, focus formation and tumorigenicity in nude mice. Although transformation was largely dependent on the presence of recombinant murine Steel Factor (SLF), the ligand to the c-Kit receptor, anchorage independent growth did occur at a low frequency in the absence of added factor, and this could not be inhibited by neutralising antibodies or by SLF anti-sense mRNA. Clones from factor-independent colonies in semi-solid agar displayed a narrow range of c-Kit surface protein levels (4.3-6.4 x 10(4) receptors/cell) which was relatively high compared with the pool from which they were derived. Analysis of a larger series of random clones derived from adherent cultures expressing different levels of c-Kit demonstrated a positive correlation between SLF-dependent, anchorage-independent growth and c-Kit protein and mRNA expression levels (respectively, Rs = 0.58, P < 0.01; and Rs = 0.53, P < 0.01) with consistent colony formation observed with clones having > 2.5 x 10(4) receptors/cell. Interestingly, two of the three clones expressing the highest levels of c-Kit protein and mRNA produced few or no colonies in the presence or absence of SLF. Sequential overexpression of human c-KIT in NIH3T3 cells using a dihydrofolate reductase (DHFR)-encoding vector and gene co-amplification through methotrexate selection, which resulted in pools expressing up to 1.5 x 10(5) receptors/cell, confirmed that high receptor densities resulted in a decrease in colony numbers. Thus, analysis of clonal and selected populations has indicated that an optimal level of c-Kit is required for transformation of NIH3T3 cells in the presence of SLF, and that some ligand-independent transformation occurs.

3T3 Cells↗

PubMed1993

Responses of the murine myeloid cell line FDC-P1 to soluble and membrane-bound forms of steel factor (SLF).

Cells of the murine interleukin-3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF) factor-dependent line, FDC-P1, express the tyrosine kinase receptor, c-kit. The ligand for c-kit, steel factor (SLF), encoded by the steel (Sl) locus, is produced as both membrane-bound and soluble forms by fibroblastoid cells. Fibroblasts derived from normal (+/+) WCB6F1 mice are known to produce both forms of SLF and were able to support FDC-P1 cells in a contact-dependent manner in the presence of neutralizing anti-GM-CSF antiserum. In contrast, Sl/Sld mutant fibroblasts, which produce only a soluble form of SLF, were incapable of supporting FDC-P1 cells in the presence of GM-CSF antiserum. These results suggested that FDC-P1 cells were being supported on fibroblast layers by membrane-bound SLF. Attempts to grow FDC-P1 cells in high levels of soluble recombinant SLF to mimic the SLF-dependent response seen in co-culture experiments showed that cells which had been previously grown in GM-CSF or IL-3 were minimally responsive to SLF at concentrations up to 100 ng/mL. Although these cultures were not supported by SLF alone, the cells showed synergistic proliferative responses to SLF combined with suboptimal levels of GM-CSF or IL-3. FDC-P1 cells could, however, be adapted to grow in SLF alone by gradual substitution of SLF for GM-CSF over a period of 3 weeks. These cells showed 5.6- to 8.4-fold and 2.5-fold higher levels of c-kit mRNA than cells grown in GM-CSF or IL-3, respectively. Downregulation of surface c-kit protein was also seen in FDC-P1 cells grown in GM-CSF or IL-3 compared with cells grown in SLF. Although FDC-P1 cells propagated in SLF were more responsive to SLF, they were still able to proliferate as well in GM-CSF and IL-3 as the cells originally grown in the latter factors. Thus, functional downregulation of c-kit by GM-CSF and IL-3 was unidirectional.

Animals↗