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E Gherardi

Publications and source records attributed to E Gherardi.

At least 19 recordsLinked to original sources
PubMed1996

An approach to random mutagenesis of DNA using mixtures of triphosphate derivatives of nucleoside analogues.

We describe a new method for random mutagenesis of DNA based on the use of a mixture of triphosphates of nucleoside analogues. The method relies on DNA amplification in vitro with Taq polymerase and in the presence of the 5'-triphosphates of 6-(2-deoxy-beta-D-ribofuranosyl)-3,4-dihydro-8H-pyrimido-[4,5-C] [1,2]oxazin-7-one(dP) and of 8-oxo-2' deoxyguanosine (8-oxodG). The newly synthesised triphosphate derivative of dP (dPTP) is an excellent substrate for Taq polymerase (Km = 22 microM versus Km = 9.5 microM for TTP); it is incorporated in place of TTP and, with a approximately fourfold lower efficiency, in place of dCTP. After 30 cycles of DNA amplification, equimolar mixtures of the four normal dNTPs and dPTP yield the following frequencies of the four transition mutations: A-->G (4.4 x 10(-2), T-->C (4.3 x 10(-2), G-->A (1.1 x 10(-2) and C-->T (1.0 x 10(-2). The triphosphate derivative of 8-oxodG (8-oxodGTP) is incorporated opposite template adenine and yields two transition mutations (A-->C and T-->G) at frequencies of 0.8 x 10(-2) and 1.2 x 10(-2) respectively. Reaction mixtures containing dPTP and 8-oxodGTP results in both dP and 8-oxodG-induced mutations and an extensive array of codon changes in the absence of insertions and deletions. The method described differs from previous mutagenesis procedures in three respects: (1) it enables very high frequencies of base substitutions (up to 1.9 x 10(-1) (2) it allows control of the mutational load via the number of DNA amplification cycles and (3) it yields both transition and transversion mutations. The procedure may find application in the generation of libraries of DNA and protein mutants from which species with improved or novel activities may be selected.

Amino Acid Sequence↗
PubMed1996

Co-expression of the HGF/SF and c-met genes during early mouse embryogenesis precedes reciprocal expression in adjacent tissues during organogenesis.

Early experiments with cells in culture and recent targeting experiments have confirmed that the mesenchyme-derived growth factor hepatocyte growth factor/scatter factor (HGF/SF) is a paracrine agent that regulates the development of several epithelial and myogenic precursor cells during organogenesis. Here, we report the expression pattern of HGF/SF and its receptor, the product of the proto-oncogene c-met, during gastrulation and early organogenesis in mouse embryo. During gastrulation, the expression of HGF/SF and c-met overlaps. Initially the two genes are expressed in the endoderm and in the mesoderm along the rostro-intermediate part of the primitive streak and, later, in the node and in the notochord. Neither HGF/SF nor c-met is expressed in the ectodermal layer throughout gastrulation. During early organogenesis, overlapping expression of HGF/SF and c-met is found in heart, condensing somites and neural crest cells. However, a second and distinct pattern of expression, characterized by the presence of the ligand in mesenchymal tissues and the receptor in the surrounding ectoderm, is seen in the bronchial arches and in the limb buds. At 13 days postcoitum (d.p.c.), only this second pattern of expression is observed in differentiated somites and several major organs (i.e., lungs, liver, and gut). The expression of the HGF/SF and c-met genes throughout embryogenesis suggests a shift from an autocrine to a paracrine signaling system. The shift takes place in early organogenesis and implies different roles of HGF/SF in development. During gastrulation, HGF/SF may affect the fate of migrating mesodermal cells and may play a role in axis determination, whereas during organogenesis, the expression patterns of HGF/SF and its receptor reflect the recently established roles in the growth of certain epithelia and the migration of specific myogenic precursor cells.

Animals↗
PubMed1996

Patients' perception of public, voluntary and private dispensaries in rural areas of Tanzania.

Eighty percent of rural dispensaries are run by the government and 19% by voluntary organisations that charge for some services. After the re-legalisation of the private health sector in 1991, private dispensaries are also emerging in villages. Privatisation is among the health reform policies of the country. Moreover, cost-sharing will be introduced at public dispensaries soon. Perception of 320 patients in the Coast Region of Tanzania on services delivered by the three health sectors has been investigated. Results show that patients are generally satisfied with the services and they would go back to the same dispensaries for treatment. Polydrug prescription was common in all sectors, while lack of prescribed drugs was a main complaint among public dispensaries patients. Voluntary dispensaries patients were less satisfied with long waiting time and with staff that did not give them enough information about the treatment. Currently, health service in public dispensaries is free but cost-sharing will be introduced soon. Most of voluntary and private dispensaries patients stated that the fees for service were moderate. The paper discusses the need for monitoring the implementation of cost sharing in public dispensaries to ensure equity in access to services by rural patients.

Adult↗
PubMed1995

The structural repertoire of the human V kappa domain.

In humans, the gene for the V kappa domain is produced by the recombination of one of 40 functional V kappa segments and one of five functional J kappa segments. We have analysed the sequences of these germline segments and of 736 rearranged V kappa genes to determine the repertoire of main chain conformations, or canonical structures, they encode. Over 96% of the sequences correspond to one of four canonical structures for the first antigen binding loop (L1) and one canonical structure for the second antigen binding loop (L2). Junctional diversity produces some variation in the length of the third antigen binding loop (L3) and in the identity of residues at the V kappa-J kappa join. However, this is limited and 70% of the rearranged sequences correspond to one of three known canonical structures for the L3 region. Furthermore, we show that the canonical structures selected during the primary response are conserved during affinity maturation: the key residues that determine the conformations of the antigen binding loops are unmutated or undergo conservative mutation. The implications of these results for immune recognition are discussed.

Amino Acid Sequence↗
PubMed1995

Scatter factor/hepatocyte growth factor is essential for liver development.

Polypeptide growth factors are important effectors of cell growth and differentiation in vitro and are thought to be critical for processes such as specification of cell fate, tissue growth and organogenesis in vivo. Scatter factor/hepatocyte growth factor (SF/HGF) is the prototype of an emerging family of growth factors that resemble in their domain structure and mechanism of activation the blood proteinase plasminogen. The cellular responses of SF/HGF are mediated by the c-Met tyrosine kinase receptor. Here we report that mice lacking SF/HGF fail to complete development and die in utero. The mutation affects the embryonic liver, which is reduced in size and shows extensive loss of parenchymal cells. In addition, development of the placenta, particularly of trophoblast cells, is impaired. Thus, SF/HGF is essential for the development of several epithelial organs.

Animals↗
PubMed1995

Measurement of the absolute number of functioning low-density lipoprotein receptors in vivo using a monoclonal antibody.

MAC188 S/S is a monoclonal antibody which can be used in vivo to measure the absolute number of functioning low-density lipoprotein (LDL) receptors in a rabbit. The antibody binds to the extra-cellular domain of the LDL receptor and binding is not blocked by the presence of LDL. When the antibody-receptor complex is internalized, receptor recycling is inhibited for several hours. Thus when saturating doses of MAC188 S/S are administered intravenously, the amount of antibody removed from the blood (minus non-specific removal) is determined solely by the total number of LDL receptors in an animal. In this study MAC188 S/S was used to measure the number of LDL receptors in control rabbits and in animals treated with 17 alpha-ethinyl oestradiol. After treatment (which caused a 47% decrease in plasma cholesterol), receptor-mediated removal of MAC188 S/S from the blood was saturated in both groups following injection of 3.0 mg of antibody per kg body weight. Based on the amount of antibody removed via the LDL receptor at this dose, the total number of accessible LDL receptors was calculated as (2.0 +/- 0.3) x 10(15) receptors per kg body weight in control rabbits and (4.0 +/- 0.4) x 10(15) receptors per kg body weight in oestrogen-treated animals. The number of receptors in various organs was also determined. The monoclonal antibody approach therefore, allows accurate determination of LDL receptor numbers in animals with markedly different concentrations of circulating LDL, conditions in which the use of endogenous ligand would be subject to significant errors.

Animals↗
PubMed1995

Universal cloning and direct sequencing of rearranged antibody V genes using C region primers, biotin-captured cDNA and one-side PCR.

Polymerase chain reaction (PCR) cloning has greatly facilitated the cloning of heavy and light chain genes from B cells and hybridomas and has been critical for the generation of natural antibody gene libraries for expression in bacteria and on filamentous phages. There remain difficulties, however, in cloning VH and VL genes from a number of mouse and rat hybridoma lines and from B cells from several other species due to insufficient sequence information. Here we describe a rapid and 'universal' strategy for cloning rearranged antibody genes from any species for which the sequence of the C segment(s) are known. First strand synthesis is primed with a biotinylated C region primer and full length cDNA is captured on streptavidin-coated magnetic beads for tailing with dGTP and terminal deoxynucleotidyl transferase. After tailing, the cDNA is captured again, amplified using polyC primers and used for direct sequencing or cloning. The use of C region primers and cDNA capture ensures that this one-side PCR procedure is efficient and rapid as well as being entirely independent of the sequence of the V segment. We demonstrate its application to the direct sequencing or cloning of the H and L chain genes from six mouse and rat hybridomas and propose that the method described will find applications in three areas: (i) cloning rearranged antibody genes in all cases in which cloning with V-J primers is not possible; (ii) repertoire studies in which an unbiased cloning procedure is required for accurate estimate of gene usage; and (iii) generation of VH and VL gene libraries from immunised animals.

Amino Acid Sequence↗
PubMed1995

Characterization of the scatter factor/hepatocyte growth factor gene promoter. Positive and negative regulatory elements direct gene expression to mesenchymal cells.

Scatter factor/hepatocyte growth factor (SF/HGF) and its receptor c-Met represent a paracrine signaling system involved in mesenchymal-epithelial interactions during development and during tumor progression. We have examined the promoters of the mouse and human SF/HGF genes by deletion mapping followed by CAT assays as well as by gel retardation and footprinting analysis. The promoter sequences are highly conserved (89.5% identity) up to position -453 from the major transcription start site but diverged considerably further upstream. Both promoters are active in mesenchymal but not epithelial cells thus reflecting the expression pattern of the SF/HGF gene in cells in vitro and in vivo. We have here identified two regulatory sequences in the SF/HGF promoter: a negative element at positions -239 to -258 and a positive element near the major transcription start site; specific deletions destroyed the activities of these elements. We were not able to localize elements on the SF/HGF promoter region that mediate the previously described effects of transforming growth factor beta, 12-O-tetradecanoylphorbol-13-acetate, and coculture of epithelial cells on SF/HGF gene expression. This study represents a first step toward understanding the intricately regulated and cell type-specific expression of the paracrine acting SF/HGF.

3T3 Cells↗
PubMed1995

Expression of HGF/SF, HGF1/MSP, and c-met suggests new functions during early chick development.

We report the cloning of fulllength cDNAs for a plasminogen-related growth factor, hepatocyte growth factor/scatter factor (HGF/SF), its tyrosine kinase receptor, c-met, and a close member of the same family, hepatocyte growth factor-like/macrophage stimulating protein (HGF1/MSP), from the chick. We have used these cDNAs to provide the first report of the expression of this family of growth factors and the c-met receptor at early stages of vertebrate development. RNAase protection and wholemount in situ hybridization were used on chick embryos between formation of the primitive streak and early organogenesis. We find patterns of expression for HGF/SF and its receptor c-met consistent with their known roles in epithelial-mesenchymal transformation and angiogenesis. In addition, these genes and HGF1/MSP are expressed in discrete locations within developing somites, suggesting a role in paraxial mesodermal development. Very strong and early expression of HGF/SF in the elevating limb buds suggests its involvement in limb outgrowth. HGF1/MSP is expressed in the notochord and then in the prospective floor plate region and could play a role in development of the neural tube. Interestingly, c-met is often more closely associated with HGF1/MSP than with its known ligand, HGF/SF, raising the possibility that c-met expression may be induced by HGF1/MSP.

Amino Acid Sequence↗
PubMed1995

HGF/SF inhibits junctional communication.

Hepatocyte growth factor/scatter factor (HGF/SF) is a fibroblast-derived protein that affects the growth, motility, and differentiation of epithelial and endothelial cells. We have investigated the effect of HGF/SF on junctional communication in mouse keratinocytes (MK cells). HGF/SF inhibited cell communication in MK cells as assessed by the transfer of a low-molecular-weight dye, Lucifer Yellow. The inhibition was rapid, the earliest effects being apparent 5 to 10 min after addition of the factor, and was transient. The decrease in dye transfer correlated with a loss of the gap junction protein connexin 43 as measured by Western blotting, probably due to increased protein degradation. The results show that junctional communication is an early target of HGF/SF activity and they are consistent with the hypothesis that gap junctions are primary targets of the action of growth factors.

Animals↗
PubMed1995

Roles of hepatocyte growth factor/scatter factor and the met receptor in the early development of the metanephros.

Several lines of evidence suggest that hepatocyte growth factor/scatter factor (HGF/SF), a soluble protein secreted by embryo fibroblasts and several fibroblast lines, may elicit morphogenesis in adjacent epithelial cells. We investigated the role of HGF/SF and its membrane receptor, the product of the c-met protooncogene, in the early development of the metanephric kidney. At the inception of the mouse metanephros at embryonic day 11, HGF/SF was expressed in the mesenchyme, while met was expressed in both the ureteric bud and the mesenchyme, as assessed by reverse transcription PCR, in situ hybridization, and immunohistochemistry. To further investigate the expression of met in renal mesenchyme, we isolated 13 conditionally immortal clonal cell lines from transgenic mice expressing a temperature-sensitive mutant of the SV-40 large T antigen. Five had the HGF/SF+/met+ phenotype and eight had the HGF/SF-/met+ phenotype. None had the HGF/SF+/met- nor the HGF/SF-/met- phenotypes. Thus the renal mesenchyme contains cells that express HGF/SF and met or met alone. When metanephric rudiments were grown in serum-free organ culture, anti-HGF/SF antibodies (a) inhibited the differentiation of metanephric mesenchymal cells into the epithelial precursors of the nephron; (b) increased cell death within the renal mesenchyme; and (c) perturbed branching morphogenesis of the ureteric bud. These data provide the first demonstration for coexpression of the HGF/SF and met genes in mesenchymal cells during embryonic development and also imply an autocrine and/or paracrine role for HGF/SF and met in the survival of the renal mesenchyme and in the mesenchymal-epithelial transition that occurs during nephrogenesis. They also confirm the postulated paracrine role of HGF/SF in the branching of the ureteric bud.

Animals↗
PubMed1995

A role for HGF/SF in neural induction and its expression in Hensen's node during gastrulation.

It was previously shown (Roberts, C., Platt, N., Streit, A., Schachner, M. and Stern, C. D. (1991) Development 112, 959-970) that grafts of Hensen's node into chick embryos enhanced and maintain expression of the L5 carbohydrate in neighbouring epiblast cells, and that antibodies against L5 inhibit neural induction by such a graft. We now show that L5 is initially widely expressed in the epiblast, but as neural induction proceeds it gradually becomes confined to and up-regulated in the early neural plate. L5 can therefore be considered as a marker for cells that are competent to respond to neural induction. We also show that Hepatocyte Growth Factor/Scatter Factor (HGF/SF) promotes the expression of L5 by extraembryonic epiblast in collagen gels after overnight culture. Explants cultured for several days in the presence of HGF/SF, as well as explants of prospective neural plate, can differentiate into cells with neuronal morphology expressing neuronal markers. To investigate whether HGF/SF is expressed in the chick embryo at appropriate stages of development, we produced specific cDNA probes and used them for in situ hybridization. We find that at the primitive streak stage, HGF/SF is expressed specifically in Hensen's node. We therefore propose that HGF/SF plays a role during the early steps of neural induction, perhaps by inducing or maintaining the competence of the epiblast to respond to neural inducing signals.

Animals↗
PubMed1994

Molecular evolution and domain structure of plasminogen-related growth factors (HGF/SF and HGF1/MSP).

Plasminogen-related growth factors, a new family of polypeptide growth factors with the basic domain organization and mechanism of activation of the blood proteinase plasminogen, include hepatocyte growth factor/scatter factor (HGF/SF), a potent effector of the growth, movement, and differentiation of epithelia and endothelia, and hepatocyte growth factor-like/macrophage stimulating protein (HGF1/MSP), an effector of macrophage chemotaxis and phagocytosis. Phylogeny of the serine proteinase domains and analysis of intron-exon boundaries and kringle sequences indicate that HGF/SF, HGF1/MSP, plasminogen, and apolipoprotein (a) have evolved from a common ancestral gene that consisted of an N-terminal domain corresponding to plasminogen activation peptide (PAP), 3 copies of the kringle domain, and a serine proteinase domain. Models of the N domains of HGF/SF, HGF1/MSP, and plasminogen, characterized by the presence of 4 conserved Cys residues forming a loop in a loop, have been modeled based on disulfide-bond constraints. There is a distinct pattern of charged and hydrophobic residues in the helix-strand-helix motif proposed for the PAP domain of HGF/SF; these may be important for receptor interaction. Three-dimensional structures of the 4 kringle and the serine proteinase domains of HGF/SF were constructed by comparative modeling using the suite of programs COMPOSER and were energy minimized. Docking of a lysine analogue indicates a putative lysine-binding pocket within kringle 2 (and possibly another in kringle 4). The models suggest a mechanism for the formation of a noncovalent HGF/SF homodimer that may be responsible for the activation of the Met receptor. These data provide evidence for the divergent evolution and structural similarity of plasminogen, HGF/SF, and HGF1/MSP, and highlight a new strategy for growth factor evolution, namely the adaptation of a proteolytic enzyme to a role in receptor activation.

Amino Acid Sequence↗
PubMed1993

Topography of apolipoprotein B in subcellular fractions of rabbit liver probed with a panel of monoclonal antibodies.

We have used a panel of anti-rabbit apolipoprotein B monoclonal antibodies in a competitive ELISA to probe the availability of apoB in rough endoplasmic reticulum, smooth endoplasmic reticulum, cis-enriched Golgi, and trans-enriched Golgi fractions from rabbit liver. The ability of each subcellular fraction to inhibit binding of monoclonal antibody to immobilized low density lipoprotein (LDL)-apoB was determined and compared with the expected inhibition based on the apoB content of the fraction. The vesicles remained closed during ELISA, demonstrated by monitoring loss of radiolabeled secretory proteins from the lumen and by measuring leakage of albumin from the vesicles. In control experiments, vesicles were permeabilized using 0.4% taurocholate. All epitopes of apoB were fully expressed in closed trans-Golgi vesicles, indicating that the membrane-bound apoB is at the cytosolic side of this fraction. In the smooth endoplasmic reticulum the epitopes were expressed between 55 and 70%, suggesting that the two pools of apoB may exist in these membranes. These results suggest that newly synthesized apoB has two possible fates. It may be incorporated into the cytosolic side of the endoplasmic reticulum from where it moves to the cytosolic side of the Golgi membrane, or newly synthesized apoB may be translocated to the lumenal surface of the endoplasmic reticulum membrane followed by assembly with lipids for secretion.

Animals↗
PubMed1993

Hepatocyte growth factor/scatter factor (HGF/SF), the c-met receptor and the behaviour of epithelial cells.

Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional protein produced by fibroblasts and other mesenchymal cells and active on epithelial and endothelial cells. The factor shares the basic domain organization of plasminogen and other blood proteases and is produced as a single-chain high molecular weight precursor which is subsequently cleaved to produce a biologically active heterodimer. HGF/SF acts on target cells through binding to a cell surface, high-affinity receptor with tyrosine kinase activity encoded by the c-met proto-oncogene. Transduction of the HGF/SF signal in epithelial cells by the met receptor leads either to: (i) cell dissociation, with loss of adhesion and junctional communication, (ii) cell division or (iii) differentiation and morphogenesis. These responses depend on target cells and culture conditions. There is preliminary evidence for a role for HGF/SF in vivo, suggesting that the factor may be involved in the early stages of embryo development and in liver regeneration. A role in cancer growth or dissemination has been proposed but remains to be clarified.

Animals↗
PubMed1993

Properties and structure-function relationship of HGF-SF.

HGF-SF is a cytokine independently isolated and characterized for its activities as a mitogen for liver cells in culture (hepatocyte growth factor, HGF) and as a factor which enhances the movement and induces the dissociation of epithelial colonies (scatter factor, SF). The factor is composed of two subunits (A and B) with a M(r) of approximately 57 K and approximately 30 K produced by proteolytic cleavage of a polypeptide precursor. Based on the complete conservation of critical cysteine residues, the domain structure of HGF-SF appears to be remarkably similar to the domain structures of plasminogen, (a blood protease involved in fibrinolysis) and the HGF-like protein (the translation product of a gene recently isolated and which shares extensive sequence identity with HGF-SF). The A subunit of HGF-SF consists of an N-terminal domain, homologous to plasminogen preactivation peptide (PAP), followed by four kringle domains. The B subunit has the basic structure of a serine protease domain although HGF-SF has no protease activity due to replacement substitutions in two of the three residues of the catalytic site. Experiments with a truncated form of HGF-SF generated by alternative splicing and with a series of deletion and point mutants generated by site-directed mutagenesis, have established that both the N-terminal and the kringle domains of the A chain and the B chain are all required for biological activity. Loss of mitogenic activity in the HGF-SF mutants is always associated with loss of motogenic activity and vice versa. Thus, the protein engineering experiments confirm the hypothesis that the mitogenic and motogenic responses to HGF-SF diverge at the post-receptor level.

Amino Acid Sequence↗