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J Ferrari

Publications and source records attributed to J Ferrari.

16 recordsLinked to original sources
PubMed1994

Cloning and expression of a soluble sialidase from Chinese hamster ovary cells: sequence alignment similarities to bacterial sialidases.

A cDNA encoding a soluble sialidase from Chinese hamster ovary (CHO) cells has been cloned and expressed. Completely degenerate oligonucleotide primers, which were based on the amino acid sequence of peptides obtained from the purified sialidase (Warner et al., Glycobiology, 3, 455-463, 1993), and the polymerase chain reaction, with single-stranded cDNA template, were employed to generate a unique oligonucleotide probe. The unique probe of 93 bp was used for screening a lambda gt 10 CHO cell cDNA library. A single clone, which contained a 1.4 kb insert, was isolated after screening 450,000 recombinants. The complete coding region of the protein, 1137 nucleotides, was contained in the isolated clone and it predicted a protein of 379 amino acids. The insert had a 186 bp 5' non-coding leader sequence and a 40 bp 3' non-coding region. No signal peptide was identified in the insert, suggesting a cytosolic localization for the protein. No significant primary sequence identities were observed when the deduced amino acid sequence of the CHO cell sialidase was compared with other mammalian proteins or microbial sialidases. However, the protein had significant sequence alignment similarity with several bacterial sialidases. Two 'Asp box' motifs in the CHO cell sialidase had a remarkable alignment positioning in the protein sequence with the similar motifs of the Salmonella LT2 and Clostridium perfringens sialidases. High levels of the enzyme were expressed in Spodoptera frugiperda cells infected with a modified Autographa californica nuclear polyhedrosis virus harbouring the sialidase cDNA.

Amino Acid Sequence↗
PubMed1991

Fetal mouse kidney maturation in vitro: coordinated influences of epidermal growth factor, transferrin and hydrocortisone.

We have investigated the individual and combined actions of epidermal growth factor (EGF), transferrin and hydrocortisone on the maturation of whole fetal mouse metanephroi maintained in serum-free conditions for up to 5 days. The presence of EGF (100 ng/ml) resulted in elevated levels of [3H]-thymidine incorporation when compared to controls; autoradiograms showed that the proliferation of mesenchymal cells in the nephrogenic zone is particularly enhanced as verified by cell counting. Brush border hydrolase activities (alkaline phosphatase and gamma-glutamyltransferase), on the other hand, were significantly diminished. Transferrin (5 micrograms/ml) slightly stimulated DNA synthesis and potentiated EGF mitogenic action. The activation of DNA replication by the growth factor seems to be mediated through the protein kinase C pathway. When added alone, hydrocortisone (10(-6) M) strongly inhibited DNA synthesis, stimulated hydrolase activities and exerted a positive effect on brush border differentiation. When combined with EGF or to EGF + transferrin, hydrocortisone counteracted the effects of these latter peptides on DNA synthesis and enzyme activities. Considering the earlier observation of a reciprocal relation between proliferation and differentiation during the neotubulogenic phase of kidney development, the results described in the current study suggest that synergistic and synarchic actions of these heterologous factors are involved in the regulation of tubulogenesis.

Animals↗
PubMed1991

Mouse fetal kidneys in serum-free organ culture: effects of epidermal growth factor and hydrocortisone.

1. The present study was undertaken to determine whether epidermal growth factor (EGF, 100 ng/ml) or hydrocortisone (HC, 10(-8)-10(-5) M) directly influence proliferation and differentiation of mouse fetal kidney maturing in serum-free organ culture. 2. Addition of EGF to the medium significantly stimulated DNA synthesis after 2 and 5 days of culture. Labelled nuclei were mainly localized in the mesenchymal tissue. Protein synthesis remained unchanged. Activities of three hydrolases, markers of brush border differentiation, were reduced. 3. Hydrocortisone (HC), at all concentrations used, significantly inhibited DNA synthesis. Labelled nuclei were distributed in various cell populations of both control and treated explants. Protein synthesis was stimulated by 10(-7) M after 5 days of culture. Hydrolase activities were slightly modified by HC treatment. 4. The present results indicate that EGF stimulates whereas HC decreases proliferation. Both factors have regulatory effects on brush border maturation. 5. Thus, this culture model is a valuable tool for the study of nephrogenesis.

Animals↗
PubMed1991

Insulin and transferrin restore important cellular functions of human fetal kidney in serum-free organ culture.

A model previously developed in our laboratory to culture human fetal kidneys in serum-free chemically defined medium was used to evaluate the direct influence of potential regulators on nephrogenesis. The aim of the present work was to verify the effects of insulin and transferrin, two hormones considered as essential in other serum-free culture systems. Explants of renal cortex from human fetuses (15-21 weeks) were cultured for 2 and 5 days in serum-free Leibovitz's L-15 medium (37 degrees C, 95% air - 5% CO2). The addition of transferrin (5 micrograms/mL) had no effect, but insulin (30, 60, and 125 mU/mL) increased DNA and protein syntheses in a dose-dependent manner. The influence of insulin (125 mU/mL) was potentiated by the addition of transferrin and the combination of the two stimulated DNA synthesis by threefold on day 2 when compared with controls and by sixfold on day 5 of culture. After 5 days, synthesis was restored to values observed at day 0. Transferrin did not modify the insulin effect on protein synthesis, since the latter was already maximally stimulated as early as day 2 of culture and at levels well above that of uncultured explants (day 0). The activities of four hydrolases considered as markers of brush border differentiation were not importantly changed by any of the hormones, supplemented alone or in combination. The results indicate that proliferation rather than differentiation is the parameter mostly influenced by these two hormones. The combination of insulin plus transferrin restores cellular functions of human fetal kidney explants cultured in serum-free medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗
PubMed1991

Synergistic influence of epidermal growth factor, insulin and transferrin on human fetal kidney in culture.

In human fetal kidneys (15-21 weeks of gestation) maintained in serum-free organ culture, protein synthesis remained relatively constant, but DNA synthesis decreased dramatically after 2 days. The addition of transferrin alone had no influence, but insulin and epidermal growth factor (EGF) both significantly stimulated DNA and protein synthesis. When supplemented in combination, transferrin strongly potentiated the insulin effect and after 5 days of culture DNA synthesis was practically restored to values observed in control uncultured renal explants (day 0). When EGF, a potent mitogen, was added as a third factor, the stimulating effectiveness of the (insulin plus transferrin) combination was significantly reduced. However, EGF had no such inhibiting influence on protein synthesis. Differentiation of brush border membranes, as evaluated by hydrolase activities, was not importantly induced nor retarded by any of the three factors supplemented either alone or in combination. The present results indicate that the individual effects of the three factors are not additive, but suggest that they rather act synergistically through a complex mechanism of receptor cross-talk. In our laboratory, there is convincing indication that the response of fetal organs varies according to age, proliferative state of tissues as well as stage of differentiation.

DNA↗
PubMed1989

Developmental profile of DNA synthesis and hydrolase activities in human fetal kidney.

This study provides original data on human fetal kidney developing during the 13th to 18th week of gestation. The parameters evaluated were DNA synthesis and the activities of 5 hydrolases which are considered as good markers of the brush border membrane differentiation. The conclusions are that DNA synthesis decreased slightly from the 16th to 18th week. The activities of maltase, trehalase, alkaline phosphatase and leucylnapthylamidase remained nearly stable during the studied period. Only the gamma-glutamyltransferase activity decreased significantly between the 15th and 16th week, then it returned close to the 13th week value. The current results suggest that during the 13-18 week period of gestation, cell proliferation is slowed down while maturation of some enzymic activities of the brush border are not importantly modified. The present basic data might be used as reference standards by investigators in the field of human nephrogenesis.

Alkaline Phosphatase↗
PubMed1988

On the presence of intermediate cells in the small intestine.

In the small intestine, the presence of transitional cells or cells intermediate between Paneth cells and goblet cells has been reported frequently for 100 years. Light microscopy and, more recently, fine structural studies have indicated that secretory granules observed in intermediate cells share some morphologic characteristics with those of granular goblet cells and of Paneth cells. In order to verify if intermediate cells in the jejunum and ileum of the adult mouse have functional similarities with either granular goblet or Paneth cells, we have studied the incorporation of sulfur-35 by radioautography and the localization of lysozyme by immunocytochemistry. After radioautography, goblet cells and, to a lesser extent, granular goblet cells had incorporated sulfur-35, whereas Paneth cells and intermediate cells were completely negative. Immunolocalization of lysozyme was done by using rabbit anti-rat lysozyme and protein A-peroxidase. After demonstration of peroxidase activity, only Paneth cells were stained and intermediate cells were negative. Therefore, intermediate cells do not contain sulfomucin or lysozyme, and they are functionally different from goblet and Paneth cells. Their function remains unknown.

Animals↗
PubMed1988

Comparison between mouse kidneys of pre- and postnatal ages maturing in vivo and in serum-free organ culture.

1. To evaluate the influence of age, DNA synthesis and brush border hydrolase activities were determined in mouse kidneys maturing in vivo and in serum-free organ culture. 2. DNA synthesis decreased with advancing age. 3. The protein content and leucylnaphthylamidase, maltase, trehalase, alkaline phosphatase and gamma-glutamyltransferase activities increased with aging. 4. The differences due to age were reproduced in kidneys maturing in culture. 5. These results show that age has a significant effect on the parameters determined, but apparently has no influence on the viability of the kidney explants in culture.

Aging↗
PubMed1988

Epidermal growth factor (EGF) influences DNA synthesis in human fetal kidneys maturing in serum-free organ culture.

Human fetal kidney explants (13-17 weeks of gestation) were maintained in serum-free organ culture. The influence of epidermal growth factor (EGF) was determined after 2 and 5 days by evaluating DNA and protein synthesis as well as the activities of five brush border hydrolases. During the studied period the overall morphology was preserved and the analysed parameters remained constant. Only DNA synthesis decreased after 2 days. The addition of EGF to the medium did not change any of the cell activities, except DNA synthesis. In fact, the incorporation of [3H]thymidine was significantly stimulated by 105% in 5-day explants cultured in the presence of the growth factor. These results indicate that EGF directly influences proliferation but not maturation of brush border enzymes in fetal human kidneys in culture.

Culture Media↗
PubMed1982

Adrenergic regulation of distention-induced gastrin release in humans.

To investigate the possible role of adrenergic nerves in neurally mediated gastrin release, we evaluated the effect of selective adrenergic blockade on the serum gastrin response to gastric distention in healthy human subjects. On separate days, 2 mg of propranolol (beta-adrenergic antagonist), 5 mg of phentolamine (alpha-adrenergic antagonist), or saline (control) was injected intravenously just before distending the stomach with 700 ml of isotonic saline. For 30 min following distention, intragastric pH was kept constant at 5.0 by in vivo titration. Propranolol reduced distention-induced gastrin release by approximately 90% (p less than 0.02), whereas phentolamine had no significant effect on the gastrin response to distention. In additional experiments, we evaluated the effect of the same doses of propranolol or phentolamine on the exaggerated gastrin response to gastric distention that occurred during cholinergic blockade with atropine. In the presence of atropine (2.3 microgram/kg i.v.), propranolol significantly (p less than 0.01) reduced distention-induced gastrin release, whereas phentolamine significantly enhanced the gastrin response to distention (p less than 0.01). We conclude that: (1) distention-induced gastrin release was reduced by propranolol, suggesting that gastric distention releases gastrin by a beta-adrenergic mechanism and (2) distention-induced gastrin release was enhanced by phentolamine, but only in the presence of atropine. Thus, adrenergic nerves appear to regulate the gastrin response to gastric distention in humans: beta-adrenergic pathways stimulate gastrin release, and alpha-adrenergic pathways may inhibit gastrin release under certain circumstances.

Adrenergic alpha-Antagonists↗
PubMed1981

Bombesin stimulates human gastric function by gastrin-dependent and independent mechanisms.

Studies were performed in six men to determine the relationship between serum gastrin concentration and gastric function during exogenous and endogenous stimulation of gastrin release. When the intragastric pH was maintained at 5.0, there were dose-related increases in gastric acid secretion with increasing serum gastrin concentrations produced either by stepwise increasing doses of intravenous gastrin or bombesin, or by intragastric perfusion with peptone. When intragastric pH was maintained at 2.5, or when intravenous atropine was given hourly, sensitivity to both exogenous and endogenously released gastrin were similarly decreased. Gastric emptying was inhibited in a dose-dependent manner by both bombesin and peptone but not by exogenous gastrin. These results suggest that bombesin and peptone stimulate gastric acid secretion through release of circulating gastrin but inhibit gastric emptying by another mechanism.

Adult↗