Fibronectin is stored but not synthesized in mature human peripheral blood granulocytes

To investigate whether peripheral blood granulocytes can synthesize the adhesive glycoprotein, fibronectin, we sought to demonstrate the presence of messenger RNA coding for fibronectin within mature circulating granulocytes. Polyadenylated-enriched RNA was isolated from human peripheral blood granulocytes, human skin fibroblasts (synthesize fibronectin) and HeLa cells (lack fibronectin) and probed with a cDNA clone coding for the cell attachment domain of fibronectin. Hybridization of a fibronectin cDNA fragment occurred with fibroblast RNA but did not occur with granulocyte RNA despite a 100 fold excess granulocyte RNA. Incubation of granulocytes with n-formyl methionyl leucyl phenylalanine, a chemotactic peptide known to augment the release of fibronectin from granulocytes, failed to induce detectable levels of mRNA for fibronectin in granulocytes. There was no difference in the quantity of fibronectin released from chemotactic peptide-stimulated granulocytes pre-incubated in the presence or absence of the protein synthesis inhibitor, cycloheximide, suggesting that fibronectin exists in a stored form in granulocytes. These data suggest that fibronectin in mature granulocytes is the product of synthesis during early myeloid maturation.     

Release of diamine oxidase into plasma by glycosaminoglycans in rats

Plasma diamine oxidase (DAO) values are enhanced by intravenous injection of heparin which releases the enzyme, synthesized in small bowel enterocytes, from binding sites located on endothelial cells of the intestinal microvasculature. Intestinal DAO, in analogy with lipoprotein lipase (another heparin-released enzyme), is believed to be electrostatically linked to endothelial binding sites composed of a glycosaminoglycan (GAG) which is presumably heparan sulphate, but the complete mechanism of enzyme release is not known. In this study we assayed in rats the DAO-releasing capability of heparan sulphate, dermatan sulphate, chondroitin sulphate A and hyaluronic acid, all heparin related compounds. Heparan sulphate, a compound with the same hexosamine as heparin but with a lower concentration of sulphated iduronic acid, induced a very high release of DAO (3-fold less than heparin), while the other tested GAGs, composed of higher proportions of non sulphated uronic acid and with galactosamine instead of glucosamine, induced a significantly lower release. In rats treated with 60 mg heparan sulphate the significant decrease in ileal mucosal DAO activity indicates that, in analogy with heparin, the high plasma enzymatic activity induced is of enterocytic origin. It is suggested that the high charge density of the compounds tested, due to the degree of sulphatation, is the decisive factor in promoting the release of intestinal DAO.     

Changes in parameters of lipoprotein metabolism during rat hepatic development

Maintenance of whole body cholesterol homeostasis is determined in part by the liver. Thus, changes in expression of hepatic parameters important in the regulation of cholesterol metabolism may play key roles in determining how homeostasis is maintained. The expression of hepatic lipoprotein uptake systems was studied during development using as a ligand very-low density lipoproteins rich in apolipoprotein E that had been obtained from hypercholesterolemic adult rats. These lipoproteins can serve as ligands for cell surface receptors recognizing apolipoproteins B and/or E. Uptake was lowest in freshly isolated fetal rat hepatocytes, increased substantially in hepatocytes from neonates and was intermediate in those from adults. Binding of these lipoproteins to liver membranes prepared from fetal, neonatal, suckling, weaned and adult rats was lowest in fetal preparations, while those from suckling, weaned and adult livers behaved similarly. Numbers of binding sites in neonatal liver membranes were similar to those in adult, but showed a different affinity. On the basis of this data, the ability of hepatocytes to recognize and remove apolipoprotein B/E-containing lipoproteins from the plasma appears to be a function of the differential expression or regulation of lipoprotein-uptake systems during development.     

Functional changes of endoplasmic reticulum membranes associated with liver regeneration

The fluidity and lipid composition of microsomal membranes have been studied at the earliest stage of liver regeneration in the rat (16 h after partial hepatectomy). The physical properties of the membranes have been measured by electron paramagnetic resonance analysis of freedom of motion of lipid and protein analogue probes. The fluidity of the hydrophobic core and of the microenvironment surrounding membrane proteins appeared to be modified, while no modifications were detectable in the fluidity at the surface or in bulk biochemical composition. The kinetic parameters of two enzymes of the endoplasmic reticulum (3-hydroxy-3-methyl glutaryl coenzyme A reductase and glucose-6-phosphatase) which are differentially localized within the membrane bilayer, were also measured. The temperature dependence of both enzymes was modified in the proliferating system, but these modifications were not consistent with the changes detectable in their specific activity. A model to explain the changes that occur in this proliferating membrane system is presented.     

Different signal transduction by epidermal growth factor may be responsible for the difference in modulation of amino acid transport between fetal and adult hepatocytes

[1-¹⁴C]-2-aminoisobutyric acid (AIB) uptake and signal transduction pattern after epidermal growth factor (EGF) stimulation were examined in freshly isolated hepatocytes from 20-day-old fetuses and 3-month-old rats. EGF induced a transient increase of AIB transport after 10 min only in adult animals; the observed unresponsiveness of fetal liver is not dependent on a lack of EGF receptors which are present though to a lesser extent on the plasma membrane in this period. As far as the production of the second messengers, inositol trisphosphate (IP₃) and calcium, is concerned, substantial differences were found: EGF increased IP₃ production in adult hepatocytes, whereas it had no effect in fetal ones. Moreover, the addition of EGF induced a calcium transient in hepatocytes from adult animals, while there was no increase in fetal cells. The lack of EGF effect on amino acid transport in fetal cells could be due to its inability to produce both IP₃ and calcium transients, suggesting that this transduction pathway is not activated during fetal life.     

Gonadotropin-releasing hormone in elasmobranch (electric ray, Torpedo marmorata) brain and plasma: chromatographic and immunological evidence for chicken GnRH II and novel molecular forms.

Gonadotropin-releasing hormone (GnRH) peptides in the brain, testis and plasma of an electric ray (Torpedo marmorata) were investigated by gel filtration chromatography, reverse phase high performance liquid chromatography and radioimmunoassay with region-specific antisera. In the brain, two major forms of GnRH were demonstrated. One form had identical chromatographic and immunological properties to chicken GnRH II, and the second, novel, molecular form had structural features in common with mammalian, chicken II and salmon GnRHs. A minor, early-eluting immunoreactive peak, possibly also a novel GnRH, was also evident. Immunoreactive GnRH was not detected in the testis. In the plasma, a single major early-eluting immunoreactive peak was demonstrated. This peak, identical to the minor peak observed in the brain, is likely to represent a novel form of GnRH which has immunological properties in common with mammalian, chicken II and salmon GnRHs. Immunoreactive GnRH was not detected in the plasma of species from other vertebrate classes, including rabbit, chicken, monitor lizard, clawed toad, frog, cichlid fish and lamprey. The finding of chicken GnRH II in a species of Chondrichthyes adds further support to our hypothesis that this widespread structural variant may represent an early-evolved and conserved form of GnRH. The presence of a GnRH molecular form in the plasma of the electric ray suggests that GnRH may reach target organs (pituitary and gonads) via the general circulation in some species of Chondrichthyes.     

Signal transduction during liver regeneration: role of insulin and vasopressin.

The relationship between cell proliferation and inositol lipid turnover has been studied by comparing the steady state of inositol derivative metabolism in quiescent and regenerating rat hepatocytes isolated at 4 h (G1 phase of first cell cycle) and 24 h (onset of M phase) after partial hepatectomy. The effect of two hormones able to regulate hepatic regeneration, insulin and vasopressin, has been considered, and the results can be summarized as follows: (i) at 4 h after partial hepatectomy, the precursor incorporation into inositol polyphosphates and the particulate phospholipase C activity increase with respect to quiescent hepatocytes, whereas the content of 11, 4, 5P3 does not change, suggesting an increased turnover of this molecule in this step of cell cycle priming; (ii) 24 h after partial hepatectomy, the radioactivity linked to IP3 and IP4, as well as soluble and particulate phospholipase C activity, and IP3 content increase, suggesting the presence, at the onset of M phase, of second messenger accumulation; (iii) only 24 h after partial hepatectomy, the inositol derivative metabolism is affected by vasopressin; and (iv) insulin exerts a modulatory role on inositol polyphosphate production without involving membrane-bound PLC activity or phosphoinositide hydrolysis. These data suggest that inositol-derived signal molecules are associated with hepatic regeneration; moreover, the metabolic pathway of such compounds seems to be regulated so that only specific inositol phosphates are present in each step of the cell cycle.     

Synergistic effects of cellulolytic and pectinolytic enzymes in degrading sugar beet pulp

Three cellulases, one hemicellulase and three pectinases were used, separately or in binary and ternary combinations, to hydrolyze dried beet-pulp, a by-product of the sugar industry. By IE-HPLC the compositions and concentrations of the sugars released were determined. The results obtained by enzymatic saccharification were compared to those obtained by acid hydrolysis. The synergistic action of cellulolytic and pectinolytic enzymes in release of total monosaccharides, and of glucose, arabinose and galacturonic acid was also studied. The combination of cellulase, hemicellulase and pectinase, commercially available, was as effective in degrading the beet pulp as the acid hydrolysis. Pectinase appeared to be the most important enzyme, since by hydrolyzing the pectic surface of the lignocellulosic substrate, it favoured the degradation of cellulose and hemicellulose by the respective enzymes.