Mitogenic activity of peptides related to the sequence of human fibroblast growth factor-1.

Linear synthetic peptides related to the human Fibroblast Growth Factor-1 (hFGF-1) segment [112-147] were tested for their capacity of mimicking FGF mitogenic activity, binding to heparin-Sepharose columns, stimulating DNA synthesis and competing with hFGF-1 for the cellular receptors. The results obtained indicated that the activity of these compounds is dependent on the presence of the sequence WFVGLK in their structures. The affinity for the cellular receptors increased when this sequence was elongated in order to incorporate amino acid residues that are important for FGF-heparin binding.

Structure and function of fibroblast growth factor.

We describe some structural requirements of the fibroblast growth factor (FGF) signaling system for the stimulation of the mitogenic response in terms of the design, synthesis and mitogenic activity of linear peptides related to the human FGF-1 sequence and the structural requirements of heparin for the potentiation of the mitogenic activity of FGF-1. The best mitogenic peptide we have synthesized so far is Ac-WFVGLKKNGSSKRGPRT-NH2, that has been shown: 1) to bind to heparin-Sepharose columns with moderate affinity, requiring about 0.5 M NaCl to be eluted from the resin; 2) to be mitogenic upon BALB/c 3T3 fibroblasts in culture (ED50 = 10-20 microM) and 3) to compete with human FGF-1 for cellular binding (ID50 = 30-50 microM). The potentiating activity of heparin upon FGF-1 has shown to be dependent on the oligosaccharide size, degree of sulfation and carboxylation. Apparently, these same requirements hold for the heparan sulfate molecules. Based on the reported studies, we propose some important requirements of an oligosaccharide to potentiate FGF-1 mitogenic activity: 1) to have a minimum of twelve units, organized as disaccharides where one of the units is a uronic acid and the second is glycosamine; 2) to have at least one iduronic acid sulfated at position 2 and 3) to have N-sulfated glycosamines, preferentially 6-O-sulfated. To have groups of negative charges is not enough: they need to be localized in a correct conformation.

Structure and function of fibroblast growth factor

We describe some structural requirements od the fibroblast growth factor (FGF) signaling system for the stimulation of the mitogenic response in terms of the design, synthesis and mitogenic activity of linear peptides related to the human FGF-1 sequence and the structural requirements of heparin for the potentiation of the mitogenic activity of FGF-1. The best mitogenic peptide we have synthesized so far is Ac-WFVGLKKNGSSKRGPRT-NH2, that has been shown: 1)to bind to heparin-Sepharose columns with moderate affinity, requiring about 0.5 M NaCl to be eluted from the resin; 2) to be mitogenic upon BALB/c 3T3 fibroblasts in culture (ED50=10-20 muM) and 3)to compete with human FGF-1 for cellular binding (ID50=30-50 muM). The potentiating activity of heparin upon FGF-1 has shown to be dependent on the oligosaccharide size, degree of sulfation and carboxylation. Apparently, these same requirements hold for the heparan sulfate molecules. Based on the reported studies, ee propose some important requirements of an oligosaccharide to potentiate FGF-1 mitogenic activity: 1) to have a minimum of twelve units, organized as disaccharides where one of the units is a uronic acid and the second is glycosamine; 2) to have at least one iduronic acid sulfated at position 2 and 3) to have N-sulfated glycosamines, preferentially 6-O-sulfated. To have groups of negative charges is not enough: they need to be localized in a correct conformation.

[The use of an automatic stapler in abdominal hysterectomies. Our experience]. / L'uso della suturatrice automatica nelle isterectomie addominali. Nostra esperienza.

One hundred and four patients were randomized into two groups: group 1 (n = 56) included patients in whom used absorbable staples in vaginal cuff closure; group 2 (n = 48) included patients with classical abdominal hysterectomy. Operating time, facility and outcome 6 months later, were evaluated. Greater costs of absorbable staples compared with sutures can readily be counterbalanced by saving allowed by shorter operating time, no peritoneal contamination, minimal tissue trauma and better cuff healing.

Long-term follow-up of Nissen fundoplication.

A study of 68 children under 7 years of age, who had had an abdominal approach Nissen operation, with a postoperative follow-up of between 4 and 14 years, is presented. The patients were classified into three groups, according to radiological appearance and cuff site. In the first group (40 patients) the cuff was intraabdominal and competent; in the second group (22 patients) the cuff was partially displaced into the thorax and was competent. 92% of the patients of these groups are currently asymptomatic and none required reoperation. In the third group (6 patients), there was disorganization of the cuff, together with its displacement into the chest, recurrence of symptoms and the authors recommended reoperation. Nissen's operation is an effective treatment for gastrooesophageal reflux in children unresponsive to medical treatment. Although displacement occurs frequently, valve competence is unaffected except in those where the cuff has disappeared and there is herniation into the thorax in which case reoperation is necessary.

Mitogenic activity of acidic fibroblast growth factor is enhanced by highly sulfated oligosaccharides derived from heparin and heparan sulfate.

The mitogenic activity of acidic fibroblast growth factor (aFGF) is potentiated by the highly sulfated hexasaccharide [IdoUA,2S-GlcNS,6S]2-[GlcUA-GlcNS,6S] the structural repetitive unit of lung heparin chains. On a mass basis, the effect of both heparin and oligosaccharide are equivalent whereas on a molar basis, heparin, which contains about seven hexasaccharide repeats, is more efficient. On the other hand, a pentasulfated tetrasaccharide or di- and tri-sulfated disaccharides are much less effective in potentiating aFGF activity than the hexasaccharide. If the growth factor is pre-incubated with the hexasaccharide at pH 7.2 and then exposed to pH 3.5 the 306/345 nm fluorescence ratio is similar to that of native aFGF indicating that the oligosaccharide stabilizes a native conformation of the protein. Heparan sulfates extracted from various mammalian tissues were also able to potentiate aFGF mitogenic activity. On a mass basis they were in general less efficient than heparin; however, heparan sulfate prepared from medium conditioned by 3T3 fibroblasts is more efficient than heparin both on a mass and molar basis. A highly sulfated oligosaccharide isolated after digestion of pancreas heparan sulfate with heparitinase I is more active than the intact molecule, reaching a potentiating effect equivalent to that of lung heparin, whereas an N-acetylated oligosaccharide isolated after nitrous acid degradation is inactive. These data suggest that the mitogenic activity of aFGF is primarily potentiated by interacting with highly sulfated regions of heparan sulfates chains.

Co-purification of proteases with basic fibroblast growth factor (FGF).

Acidic and basic fibroblast growth factors (FGFs) are proteins of 16-18 kDa. Other forms of 25-30 kDa related to this growth factor family have recently been described. All these components bind tightly to heparin-Sepharose, a property that allows the purification of several FGF-related proteins. During the purification of acidic and basic FGFs from bovine pituitary glands, we detected the presence of 28-30 kDa components that are immunoreactive against anti-basic FGF antisera. However, microsequencing analysis revealed that the 28-30 kDa components are lysosomal proteases that co-elute with basic FGF from heparin-Sepharose columns. The involvement of these proteases in the etiology of microheterogenous forms of FGFs and/or release of FGFs from the extracellular matrix is discussed.

Pituitary fibroblast growth factors: immunocharacterization of an acid component and N-terminal sequence analysis of a truncated basic component.

Extraction of bovine pituitaries at pH 7.0, in the presence or absence of protease inhibitors (PMSF, leupeptin, pepstatin A and EDTA) yielded both basic and acidic FGF components that were characterized by Western blotting and sequence analysis. Basic FGF comprised several components: an 18 KDa form that is similar, if not identical, to the basic FGF (1-146) already described; a 17 KDa form that is likely to be a new truncated molecular species (11-146) and a group of immunoreactive components of about 29 KDa. Acidic FGF showed several active components of pI 4.5-6.5. The most active component has a pI of approximately 5.0; molecular weight of 17 KDa and is shown, by Western blotting, to be similar to a truncated form of bovine brain acidic FGF. The biological activity of the latter component is shown to be neutralized by anti-brain acidic FGF antiserum.

Bovine pituitary heparin-binding fibroblast growth factors: acidic and basic forms.

1. Bovine pituitaries contain both acidic and basic fibroblast growth factor (FGF). Both forms can be prepared by heparin-affinity chromatography. The complete separation of acidic FGF from basic contaminants requires an additional isoelectric focusing step. In contrast, heparin-affinity chromatography provides basic FGF free of acidic FGF. 2. Acidic FGF was characterized by its isoelectric point (4.5-6.4) molecular weight (15-18 kDa), chromatographic behavior and reaction with antisera specific for brain acidic FGF. The results suggest that there is a close relationship between pituitary and brain acidic FGF. They also confirm our previous results obtained by preparing acidic FGF through several isoelectric focusing steps and establish that bovine pituitary indeed contains acidic FGF. 3. Basic FGF was characterized by its molecular weight (15-17 kDa), pI (9.0-10), chromatographic behavior, electrophoretic mobility and reaction with specific antiserum. It seems to be similar to the material already described by other groups.

Neuropeptide-metabolizing peptidases in neuro-2a neuroblastoma and C6 glioma cells.

Mouse Neuro-2a neuroblastoma and rat C6 glioma cloned cells were screened for neuropeptide-metabolizing peptidases using a kininase bioassay combined with a time-course bradykinin-product analysis, and a fluorimetric assay for prolyl endopeptidase. The complementary peptide products Arg1----Phe5/Ser6----Arg9 and Arg1----Pro7/Phe8-Arg9 were released during bradykinin (Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9) inactivation by homogenates of Neuro-2a and C6 cells. The 1:1 stoichiometry of the complementary fragments and their high yields, at 10% bradykinin inactivation, demonstrated the sites of hydrolysis. The initial rate of Phe5-Ser6 bond cleavage was six-fold higher than that of the Pro7-Phe8 bond. These sites of cleavage can be attributed to enzymes similar to endopeptidase A (Phe5-Ser6) and prolyl endopeptidase (Pro7-Phe8) on the basis of the specificity and sensitivity to inhibitors of the kininase activity in Neuro-2a and C6 cell homogenates. Kininase and prolyl endopeptidase specific activities (fmol/min/cell) were 10.5 and 12.4 for Neuro-2a, and 1.5 and 2 for C6 homogenate, respectively. The recovery of kininase activity was 2.2-fold higher in the particulate than in the soluble (105,000 g for 1 h) neuronal fraction, whereas the amount of prolyl endopeptidase activity was about the same in both fractions. Kininase and prolyl endopeptidase activities in C6 cells were recovered mostly in the soluble fraction. Prolyl endopeptidase specific activity decreased 10-fold in serum-starved Neuro-2a cultured cells, with no change in activity in similarly treated C6 cells. In contrast, kininase specific activity in both cell types was essentially unaffected on serum-deprivation-induced differentiation.

Purification and partial characterization of an acidic fibroblast growth factor from bovine pituitary.

Isoelectric focusing has allowed us to fractionate pituitary extracts into basic (pI 8-9) and acidic (pI 4-5) fibroblast growth factor. The acidic fibroblast growth factor (a) is stable upon refocusing, (b) migrates as an acidic protein in urea-containing gel electrophoresis; (c) is not cell-specific, being active with fibroblasts, adrenal, and glial cells, and (d) is a heterogeneous protein fraction with active components of different pI values. The component of pI 4.7, purified to or near homogeneity by isoelectric focusing shows a single peak of activity (Mr = 12,000) in gel chromatography and a single protein band of apparent Mr = 15,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Maximal restimulation of DNA synthesis initiation on serum-deprived 3T3 fibroblasts is achieved at 1-2 ng/ml; activity with rat glial cells (C6-3D) is less pronounced than with 3T3 fibroblasts.

[Congenital vesico-vaginal fistula (author's transl)]. / Fístua vesicovaginal congénita.

The case of a girl of five years with a congenital vesico-vaginal fistula derived from a persistent left mesonephric duct (or Wolffran duct) is described. The only associated malformation found in this patient was a left renal agenesis.

Pituitary fibroblast growth factors: partial purification and characterization.

1. Isoelectric focusing of fractions derived from pH 7.0 extracts of bovine pituitary glands has been used to separate acidic and basic fibroblast growth factor (FGD) activity. FGF activity was assayed by measuring the initiation of DNA synthesis in Swiss mouse 3T3 fibroblasts. 2. Acidic and basic FGF activity present in crude extracts were not separated by chromatography on CM-Sephadex presumably because of association between acidic FGF and basic proteins. 3. Isoelectric focusing was used to separate acidic (pl 4-5) and basic (pI 8-9)FGF. The activity of acidic FGF was stable in urea and beta-mercaptoethanol, whereas basic FGF was inactivated. The activity of acidic FGF precipitated at pH 4.5 and was recovered when the precipitate was solubilized at pH 8, whereas basic FGF was soluble and stable at pH 4.5. 4. Acidic FGF at 10 ng/ml was as active as 1% fetal calf serum for the initiation of DNA synthesis. This potency is comparable with homogeneous preparations of basic FGF described in the literature.

Malignant tumor emboli to the peripheral vessels.

The first case of tumor emboli trapped in a popliteal aneurysm and the longest survivor for this condition are reported. The characteristics of 25 cases reported in the literature since 1940 and the mechanism of tumor spread are reviewed. Tumor emboli represent the late stage of a malignancy and treatment does not seem to affect long-term survival.

On the regulation of DNA synthesis in a line of adrenocortical tumor cells: effect of serum, adrenocorticotropin and pituitary factors.

Y-1 adrenal cells responded to serum step down by a several fold decrease in DNA synthesis. Serum starved cells resumed DNA synthesis upon serum step up. ACTH and cAMP inhibited DNA synthesis both at low and high serum concentrations, a fact previously known. Pituitary, brain and liver crude extracts stimulated DNA synthesis in serum starved cells. Purified pituitary factors preparations contained two activities: one specific for Y-1 cells and another active with both fibroblasts and Y-1 cells. The kinetics of restimulation of DNA synthesis by serum and pituitary factors was studied. DNA synthesis restimulation occurred after a lag of 11 hours. This lag did not vary irrespective of the type of stimulator or its concentration. Cells entered S phase continuously at a rate which increased with increasing concentrations of the stimulator. Cells became refractory to the inhibitory action of ACTH five hours before entering S phase. The implications of these data to the understanding of cell growth control are considered.

Protein factors regulating mammalian cell growth.

The preparation of active protein or proteins extracted from pituitary glands is called PF (pituitary factor). PF stimulates initiation of growth in different types of cells: 3T3 mouse fibroblast WI-38 human fibroblasts, Y-1 mouse adrenal cells and C-6 rat glial cells. Proteases like trypsin and thrombin showed no growth promoting activity under the conditions of PF assay. PMSF (phenilmethyl sulfonyl fluoride) treatment did not inactivate PF. No protease activity was detected in PF using BAPNA, azocasein and azoalbumin as substrates. It is then suggested that PF growth promoting activity is not due to proteases present in the preparation. Preliminary results are presented indicating that a functional rat pituitary cell line (GH3) might secrete a growth factor in culture.

Induction of rho-minus mutants in Saccharomyces cerevisiae by guanidine hydrochloride. I. Genetic analysis.

Guanidine hydrochloride (GuHCl) induced in Saccharomyces cerevisiae cytoplasmic petite mutants (rho-) of the suppressive type. However, it was unable to induce the neutral type, even after prolonged incubation or increased drug concentration. No correlation was found between the degree of suppressiveness and the time of incubation of yeast cells with guanidine hydrochloride. The suppressiveness of rho- induced was not altered by further treatment with GuHCl, whereas it was reduced upon treatment with ethidium bromide (EtBr). Some mitochondrial genetic information was lacking in the rho- mutants induced by GuHCl, as demonstrated by the loss of the gene for erythromycin resistance and by reduced buoyant density of mitochondrial DNA of some rho-. There was no correlation between the degree of suppressiveness of the rho- induced by GuHCl and the buoyant density of the mutant mitochondrial DNA.