Endothelial cells require functional FLVCR1a during developmental and adult angiogenesis.

The Feline Leukemia Virus Subgroup C Receptor 1a (FLVCR1a) is a transmembrane heme exporter essential for embryonic vascular development. However, the exact role of FLVCR1a during blood vessel development remains largely undefined. Here, we show that FLVCR1a is highly expressed in angiogenic endothelial cells (ECs) compared to quiescent ECs. Consistently, ECs lacking FLVCR1a give rise to structurally and functionally abnormal vascular networks in multiple models of developmental and pathologic angiogenesis. Firstly, zebrafish embryos without FLVCR1a displayed defective intersegmental vessels formation. Furthermore, endothelial-specific Flvcr1a targeting in mice led to a reduced radial expansion of the retinal vasculature associated to decreased EC proliferation. Moreover, Flvcr1a null retinas showed defective vascular organization and loose attachment of pericytes. Finally, adult neo-angiogenesis is severely affected in murine models of tumor angiogenesis. Tumor blood vessels lacking Flvcr1a were disorganized and dysfunctional. Collectively, our results demonstrate the critical role of FLVCR1a as a regulator of developmental and pathological angiogenesis identifying FLVCR1a as a potential therapeutic target in human diseases characterized by aberrant neovascularization.

Hemoglobin - a novel ligand of hepatocyte ectopic F1-ATPase.

The liver is largely responsible for free hemoglobin uptake, but the molecular mechanism of this phenomenon has never been revealed. This paper presents the results of the study on hemoglobin binding components of the hepatocyte membrane that were purified using affinity chromatography on a hemoglobin matrix and identified by peptide mass fingerprinting. Both F1-ATPase alpha and beta subunits were retrieved. The binding was confirmed via an intrinsic fluorescence quenching study using a purified recombinant F1-ATPase beta subunit, and the dissociation constant for the complex was estimated from the saturation binding curve (Kd = 7.5 x 10(-7) M). The results indicate that haemoglobin binds to hepatocyte ectopic F1-ATPase. We suggested the plausible role of the receptor in endocytosis of haemoglobin by the hepatocyte.

Alumina-zirconia composites functionalized with laminin-1 and laminin-5 for dentistry: effect of protein adsorption on cellular response.

The present paper describes a study on laminin interaction with the surface of two alumina-zirconia composites with different percentages of ZrO2, both with submicrometric grain size. As major molecules within the basement membrane (BM), laminins are important protein fragments for epithelial cell adhesion and migration. On the other hand, alumina-zirconia composites are very attractive materials for dental applications due to their esthetic and mechanical properties. X-Ray photoelectron spectroscopy and atomic force microscopy were used to study the adsorption of two types of laminin, laminin-1 (Ln-1) and laminin-5 (Ln-5), onto the ceramics surfaces. The in vitro cell response was determined by intracellular phosphorylation of major kinases. Ceramics samples functionalized with laminins showed better cellular activation than untreated specimens; furthermore, cellular activation was found to be greater for the composite with higher percentage in zirconia when functionalized with Ln-5, whereas the adsorption of Ln-1 resulted in a greater activation for the alumina-rich oxide.

Analysis of the murine phosphoinositide 3-kinase gamma gene.

Phosphoinositide 3-kinase gamma is preferentially expressed in leukocytes. PI3Kgamma is activated by betagamma subunits of heterotrimeric G-proteins, which thus link seven transmembrane helix receptor activation to phosphatidylinositol (3,4,5)-trisphosphate production. Here we describe the molecular cloning of the murine PI3Kgamma cDNA, the PI3Kgamma gene structure, its chromosomal assignment and the analysis of promoter activity. The mouse cDNA shares 86% identity to its pig and human orthologues at the nucleotide level. The MmPI3Kgamma gene spans approximately 30kb and comprises 11 exons. RACE-PCR indicated the presence of multiple start sites generating 5' UTRs with different lengths, the longest being 874bp. The putative promoter region contains no TATA box but several putative binding sites for hematopoietic specific transcription factors. A 1200bp long sequence upstream the first transcription start site was found to possess tissue specific promoter activity. Deletion constructs revealed two contiguous regions, with activator function, ranging from positions -139 to -557, and with inhibitory function, ranging from positions -557 to -892. FISH analysis revealed that the MmPI3Kgamma is located on chromosome 12 band B and that the human orthologue is positioned on chromosome 7q22.2-22.3. In spite of some differences in the ATP-binding site, recombinant murine PI3Kgamma protein is equally sensitive to wortmannin as its human counterpart. This suggests that mouse models will provide reliable results in the assessments of novel PI3Kgamma inhibitors.

Defective recovery and severe renal damage after acute hemolysis in hemopexin-deficient mice.

Hemopexin (Hx) is a plasma glycoprotein mainly expressed in liver and, less abundantly, in the central and peripheral nervous systems. Hx has a high binding affinity with heme and is considered to be a major transport vehicle of heme into the liver, thus preventing both heme-catalyzed oxidative damage and heme-bound iron loss. To determine the physiologic relevance of heme-Hx complex formation, Hx-deficient mice were generated by homologous recombination in embryonic stem (ES) cells. The Hx-deficient mice were viable and fertile. Their plasma iron level and blood parameters were comparable to those of control mice and they showed no evidence of tissue lesions caused by oxidative damage or abnormal iron deposits. Moreover, they were sensitive to acute hemolysis, as are wild-type mice. Nevertheless, Hx-null mice recovered more slowly after hemolysis and were seen to have more severe renal damage than controls. After hemolytic stimulus, Hx-deficient mice presented prolonged hemoglobinuria with a higher kidney iron load and higher lipid peroxidation than control mice. Moreover, Hx-null mice showed altered posthemolysis haptoglobin (Hp) turnover in as much as Hp persisted in the circulation after hemolytic stimulus. These data indicate that, although Hx is not crucial either for iron metabolism or as a protection against oxidative stress under physiologic conditions, it does play an important protective role after hemolytic processes.

The murine Y1 receptor 5' upstream sequence directs cell-specific and developmentally regulated LacZ expression in transgenic mice CNS.

The Y1 receptor for neuropeptide Y (NPY) is highly expressed in mammalian CNS where it mediates the activation of several neurobiological functions. We have previously demonstrated that a 1.3-kb fragment upstream of the transcription initiation sites of the murine Y1 receptor gene is able to direct specific expression of reporter genes in neuronal cell cultures. In the present study transgenic mice harbouring this putative promoter region linked to the LacZ reporter gene were generated and analysed for temporal and spatial distribution. Ten transgenic lines expressed beta-galactosidase in the CNS but not in other organs such as heart, liver and kidney. Histochemical analysis of brain from adult transgenic mice showed specific expression of the transgene in specific brain regions with little variation. Four transgenic lines showed characteristic patterns of beta-galactosidase activity in the brain that are consistent with the expression of the endogenous gene. Prominent LacZ activity was present in several telencephalic and diencephalic structures, including deeper layers of cerebral cortex, amygdaloid complex, hippocampus, preoptic area, several thalamic and hypothalamic nuclei and habenula. The ontogeny analysis indicates that the LacZ expression agrees with the temporal expression pattern of rat Y1 receptor mRNA. These data demonstrate that the 1.3-kb upstream region of the murine Y1 receptor gene contains the cis acting elements required for establishing a CNS-restricted and developmental stage-specific pattern of expression in vivo. Moreover they provide further information on the distribution of this NPY subtype receptor in mammalian brain.

Green fluorescent protein as a reporter of gene expression in transgenic mice.

We used the green fluorescent protein (GFP) from the jellyfish Aequorea victoria as a reporter of gene expression in transgenic mice. The GFP coding sequence was placed under the control of the human hemopexin and the mouse beta1 integrin promoter that were previously studied in transgenic mice using the lacZ reporter gene. We showed that GFP has a higher degree of sensitivity compared to the lacZ reporter gene allowing to identify cells with low and otherwise undetectable beta-galactosidase activity. Thus we showed the potentiality of GFP in replacing lacZ as a reporter gene to investigate promoter mapping and gene regulation in transgenic mice.

Ciliary neurotrophic factor constitutively expressed in the nervous system of transgenic mice protects embryonic dorsal root ganglion neurons from apoptosis.

Ciliary neurotrophic factor (CNTF) is a potent survival factor for several neuronal populations. It is expressed postnatally by Schwann cells in the peripheral nervous system and by some glial and neuronal cells in the central nervous system. We used the promoter of the neurofilament light chain gene to produce transgenic mice that express CNTF in neurons from the beginning of neuronal differentiation. These transgenic animals may represent a suitable model to identify neuronal cell types responsive to CNTF in vivo and to study the mechanism of action of this neurotrophic factor. We show that dorsal root ganglion neurons of transgenic mice expressing CNTF in neurons are protected from apoptosis during embryonic development: 40% of these cells undergo apoptosis between embryonic day 12.5 and postnatal day 5 in transgenic mice whereas 60% do so in control animals. However, protection from apoptosis does not result in an increase in the total number of neurons at the end of development. We discuss our results with regard to CNTF potentialities in vivo and the significance of programmed cell death during development.

Specific expression in brain and liver driven by the hemopexin promoter in transgenic mice.

Transgenic mice harboring the human hemopexin promoter sequences linked to the lacZ reporter gene were generated and analyzed for temporal and spatial distribution of beta-galactosidase. Upstream sequences spanning from -1800, -700 and -500 bp to the transcription start point direct regulated beta-galactosidase expression specifically to the liver and to the brain of transgenic mice. These results suggest that the 500 bp DNA fragment flanking the 5'end of the human hemopexin gene contains the cis-acting elements required for tissue and developmental stage-specific expression in vivo and provide evidence for a new extrahepatic site of expression of the hemopexin gene.

Analysis of regulatory regions of the ciliary neutrophic factor gene in transgenic mice.

In order to study the regulatory regions of the human ciliary neurotrophic factor (CNTF) gene we made constructs containing sequences upstream and downstream of CNTF coding regions and the lacZ gene and analysed their expression in transgenic mice. We show that 240 bp upstream of the translation start codon are sufficient for the transcription of the lacZ gene. A further 4 kb upstream sequence is required for the expression of the transgene in Schwann cells. These two upstream regions together with a 2 kb downstream fragment drive high level of expression of the lacZ gene in the sciatic nerve. Our results indicate that these three fragments contain regulatory regions able to mimic the CNTF expression pattern in the mouse peripheral nervous system.

In vitro study of olfactory receptor neurones expressing the dipeptide carnosine.

Olfactory neuroepithelial (OE) cells were dissociated from late stage embryonic mice and analysed for carnosine expression. The yield of carnosine neurones was twice as high when the OE cells were seeded along with the olfactory bulb cells. Carnosine neurones resulted from both in vitro survival and neurogenesis, and were associated with clusters of underlying flat cells immunopositive for keratin. Our results demonstrate that olfactory neurones expressing their neurotransmitter carnosine can be studied in culture, and the close association with keratin-immunopositive basal cells suggests that they are dependent on these cells for survival and/or differentiation.

The use of radioactively labelled riboprobes for in situ hybridization: background and examples of application.

An overview is provided concerning the preparation and use of radiolabelled single-stranded RNA probes (riboprobes) for in situ hybridization. All theoretical aspects are covered as well as some practical examples from the authors' experience. The use of riboprobes for in situ hybridization represents a valid and highly sensitive alternative to oligonucleotide probes. A sample protocol is finally described in detail: this should enable any investigators to design the ISH assay that best fits their particular needs.

Cloning and expression of human ciliary neurotrophic factor.

Ciliary neurotrophic factor (CNTF) is a survival factor for avian ciliary ganglion neurons and a variety of other neuronal cell types in vitro. We report here the cloning of the entire genomic sequence encoding human CNTF and its primary structure. Biologically active CNTF has been expressed in Chinese hamster ovary cells from a human genomic DNA clone. Human CNTF has no significant sequence similarity to any previously reported protein, although approximately 84% similarity exists compared with rat and rabbit CNTF. The lack of both an N-terminal signal sequence and consensus sequences for glycosylation or hydrophobic regions, and the fact that active CNTF is expressed but not released into the culture medium of transfected cells, argue in favour of human CNTF as a cytosolic protein. These data provide a basis for understanding the role of CNTF in nervous system physiology and pathology.