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L Servillo

Publications and source records attributed to L Servillo.

16 recordsLinked to original sources
PubMed1992

Porins and lipopolysaccharide stimulate platelet activating factor synthesis by human mesangial cells.

Porins, a family of hydrophobic proteins located in the outer membrane of the cell wall of gram-negative bacteria and lipopolysaccharide (LPS), were shown to stimulate the synthesis of platelet activating factor (PAF), a phospholipid mediator of inflammation and endotoxic shock, by cultured human glomerular mesangial cells (MC). The synthesis of PAF induced by porins was rapid (peak at 20 min) and independent either from contamination by LPS or from generation of an endotoxin-induced cytokine such as tumor necrosis factor (TNF) since it was not prevented by cycloheximide, an inhibitor of protein synthesis or anti-TNF blocking antibodies. LPS also stimulated PAF synthesis by MC. However, the kinetic of PAF synthesis induced by LPS was biphasic with an early and transient peak at 10 minutes and a second and sustained peak at three to six hours. This second peak required an intact protein synthesis and was prevented by anti-TNF antibodies, suggesting the dependency on LPS-induced synthesis of TNF. Experiments with labeled precursors demonstrated that in MC, either after stimulation with porins or LPS, PAF was synthesized via the remodeling pathway that involves acetylation of 1-0-alkyl-sn-glyceryl-3-phosphorylcholine (2-lyso-PAF) generated from 1-0-alkyl-2-acyl-sn-glyceryl-3-phosphorylcholine by phospholipase A2 (PLA2) activity. Porins and LPS, indeed, induced PLA2-dependent mobilization of [14C]-arachidonic acid that was inhibited by p-bromodiphenacylbromide (PBDB). PBDB, an inhibitor of PLA2, also blocked PAF synthesis by preventing the mobilization of 2-lyso-PAF, the substrate for PAF-specific acetyltransferase.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Outer Membrane Proteins↗
PubMed1992

Platelet-activating factor biosynthesis by cultured mesangial cells is modulated by proteinase inhibitors.

Rat mesangial cells stimulated with calcium ionophore A23187 and phagocytosis were shown to produce platelet-activating factor (PAF), a mediator of inflammation and endotoxic shock. In the study presented here, the cultured human mesangial but not epithelial cells synthetized PAF not only in response to calcium ionophore A23187 and phagocytosis of immunoglobulin G-coated latex beads, but also after stimulation with cytokines such as tumor necrosis factor-alpha and interleukin-1 beta. PAF synthetized after stimulation with A23187 and to a lesser extent with phagocytosis was partially released. In contrast, PAF synthesized by stimulation with tumor necrosis factor-alpha and interleukin-1 beta remained cell associated. Experiments with labeled precursors demonstrated that PAF was synthetized via the remodeling pathway that involves the activation of phospholipase A2 and of an acetyl-coenzymeA:2-lyso-PAF acetyltransferase. Synthetic inhibitors of serine proteases as well as plasma alpha 1-proteinase inhibitor inhibited the activation of phospholipase A2 detected as release of (14C) arachidonic acid and the activation of acetyl-CoA:2-lyso-PAF acetyltransferase at concentrations 100-fold lower than those present in plasma. This raises the question about the ability of mesangial cells to synthetize PAF in vivo. However, the inhibitory effect of plasma alpha 1-proteinase inhibitor may be abrogated by oxidative inactivation due to a concomitant stimulation of mesangial cell respiratory burst or in zones of close contact among cells or matrix, which have been shown to exclude antiproteinases.

Acetyltransferases↗
PubMed1991

Arachidonic acid metabolites and chloride secretion in rabbit distal colonic mucosa.

The relationships between arachidonic acid (AA) metabolism and chloride secretion were investigated in mucosal preparations of rabbit distal colon. Tissues displayed a significant cyclooxygenase activity already in nonstimulated conditions and incubation with exogenous AA and calcium ionophore A23187 produced a predominant prostaglandin F2 alpha (PGF2 alpha) profile [PGF2 alpha greater than PGE2 greater than thromboxane B2 (TxB2) greater than 6-keto-PGF1 alpha] as assessed by HPLC of tissue homogenates, whereas 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) was not detected in AA- or A23187-stimulated tissues. Radioimmunological assays showed that PGE2 synthesis was time dependent, plateaued at 10 min, and proceeded at rates 15-20 times over TxB2 and 6-keto-PGF1 alpha. Among the PGs produced by colonic mucosa, only PGE2 and, to a lower extent, PGF2 alpha were found to stimulate chloride secretion and cAMP synthesis. Pretreatment with 10 microM 5,8,11,14-eicosatetraynoic acid, a cyclo- and lipoxygenase inhibitor, prevented AA-induced chloride secretion and PG and cAMP synthesis with the same strength as the cyclooxygenase inhibitor indomethacin. No effects were found after preincubation with nordihydroguaiaretic acid, a lipoxygenase blocker with moderate cyclooxygenase inhibitory properties, and caffeic acid, a lipoxygenase inhibitor. 5-HETE (5 microM) had no effect on short-circuit currents (Isc) and chloride transport, but it significantly reduced the increase in Isc, chloride secretion, and PGE2 synthesis elicited by AA or A23187. Platelet-activating factor, reported to stimulate rabbit colon Isc through an indomethacin-sensitive pathway, was not detected at concentrations as low as 10(-10) M.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1991

Mechanism of arachidonic acid transport across rabbit distal colonic mucosa.

The initial rate of [1-14C]arachidonic acid (AA) entry in the serosal side of rabbit distal colonic mucosa mounted in Ussing-type chambers is linear and independent of intracellular metabolism. When the maximal AA uptake was plotted as a function of medium AA concentration in ranges between 50 and 500 nM, saturation of the AA uptake with increasing concentrations was observed. The time course of the uptake of oleic acid and palmitic acid was similar to that observed with AA, and their separate addition to incubation medium strongly reduced the AA uptake. The influx of arachidonate was largely inhibited by ouabain and by incubation with mucosal sodium-free solution and amiloride, while it was increased when colonic mucosa was exposed to luminal amphotericin B. However, voltage-clamp studies showed that the AA entry rate appeared to be linearly related (r = 0.99) to transepithelial potential difference (PD) and suggested that the sodium dependence of AA translocation is an indirect effect of the changes in transepithelial PD induced by sodium transport shifts. These features provide evidence that there is a common entry pathway for AA and other long-chain free fatty acids mediated by a mechanism of facilitated diffusion driven by transmembrane PD.

5,8,11,14-Eicosatetraynoic Acid↗
PubMed1990

A glycoprotein inhibitor of pectin methylesterase in kiwi fruit (Actinidia chinensis).

The finding of a powerful inhibitor of pectin methylesterase in ripe kiwi fruit is reported. The inhibitor was revealed to be a glycoprotein. It was purified to homogeneity and found to have a molecular mass of about 28 kDa, as estimated by gel filtration chromatography, SDS/PAGE and analytical ultracentrifugation. The sugar portion is composed of galactose, arabinose and rhamnose, the latter being much less represented. The amino acid composition showed a very high content of acidic residues compared to basic ones, which is the reason for the very low isoelectric point of the protein (less than 3.5). The kind of inhibition on kiwi pectin methylesterase was found to be competitive with an apparent Ki of 0.22 microM, using citrus pectin as a substrate. Moreover, the inhibitor is effective in inhibiting pectin methylesterase in the pH range 3.5-7.5. Kiwi inhibitor appears to be specific for pectin methylesterase, inasmuch as it was found to be ineffective against other polysaccharide-degrading enzymes, such as polygalacturonase and amylase. Conversely, it appears to be completely aspecific as far as the pectin methylesterase source is concerned. In fact, it was found to inhibit this enzyme effectively from all the sources we assayed, i.e. orange, tomato, apple, banana, potato.

Amino Acids↗
PubMed1989

Conformation and reactivity changes induced by N-methylkirromycin (aurodox) in elongation factor Tu.

Kirromycin and related antibiotics inhibit protein synthesis in bacteria by acting on elongation factor Tu (EF-Tu). We have studied the effects of N-methylkirromycin (aurodox) on some molecular properties of this protein. The binding of the antibiotic causes a dramatic variation in the protein fluorescence emission spectrum with the appearance of a new maximum at around 340 nm. Addition of aurodox to trypsinized EF-Tu resulted in an emission spectrum similar to that of the denatured intact factor. Fluorescence lifetime analysis performed by a multifrequency phase fluorometer indicated that the fluorescence emission of the factor is heterogeneous with the major component having a lifetime near 4.8 ns in the absence and 6.6 ns in the presence of the antibiotic. These results were interpreted in terms of an antibiotic-induced environmental modification of the unique tryptophan residue of the protein leading to an increase in its quantum yield. However, aurodox did not modify the solvent exposure of this residue, as judged by fluorescence quenching experiments. Moreover, 1-anilino-8-naphthalenesulfonate (ANS) binding studies, as well as analysis of the protein reactivity toward the sulfhydryl group reagent 5,5'-dithiobis(2-nitrobenzoate) (DTNB), showed that, in the presence of aurodox, the behavior of the EF-Tu-GDP complex nears that of EF-Tu.GTP. These results strongly support the hypothesis that aurodox not only confers a "GTP-like" conformation to the EF-Tu.GDP complex but also produces a less stable folding of the protein around the tryptophan residue that may contribute to the multiple functional effects of this antibiotic.

Anti-Bacterial Agents↗
PubMed1988

Evidence of a yeast proteinase specific for elongation factor 2.

Two proteinases active on elongation factor 2 have been found in yeast. The former hydrolyzes the factor producing a single ADP-ribosylatable fragment, whereas it does not produce any fragment when incubated with different proteins. The latter, less specific, is active in cleaving both EF-2 and other proteins giving rise to a noticeable number of fragments. Moreover, when native EF-2 is incubated with the most specific of the two proteinases, the amount of the ADP-ribosylatable fragment increases with time, while no fragments are evident when ADP-ribosylation of EF-2 comes before its incubation with the proteolytic enzyme. A possible regulatory role of this proteinase on EF-2 turnover is hypothesized.

Adenosine Diphosphate Ribose↗
PubMed1985

1-N6-Etheno-ADP-ribosylation of elongation factor-2 by diphtheria toxin.

Diphtheria toxin fragment A is able to inhibit protein synthesis in the eukaryotic cell by ADP-ribosylating the diphthamide residue of elongation factor-2 (EF-2) [(1980) J. Biol. Chem. 255, 10710-10720]. The reaction requires NAD as ADP-ribose donor. This work reports on the capacity of an NAD analog, the nicotinamide 1-N6-ethenoadenine dinucleotide (epsilon NAD), to be a substrate of diphtheria toxin fragment A in the transferring reaction of the fluorescent moiety, the epsilon ADP-ribose, to the EF-2. As a consequence of the transfer of the epsilon ADP-ribosyl moiety to the EF-2, there is an increase in the emission intensity of the fluorophore and a blue shift in its emission maximum. The epsilon ADP-ribosylated EF-2, like ADP-ribosylated EF-2, retains the capacity to bind GTP and ribosome. The utility of introducing a fluorescent probe in a well defined point of the EF-2 molecule for conformational or binding studies is discussed.

Adenosine Diphosphate↗
PubMed1979

Histidine degradation enzymes in rat liver: induction by high protein intake.

High protein dietary content stimulates urea formation in ureotelic animals but does not exert almost any effect on ammonia production from L-amino acids in vitro. L-histidine and L-threonine are the only amino acids which are most actively deaminated by ureotelic animals fed on a high protein diet. All the steps of L-histidine metabolism have been studied: it has been found that both the histidine transaminase pathway and the histidase pathway are stimulated. Glutamic acid is also a product of histidine catabolism through the histidase pathway, but its catabolism is unaffected by the dietary protein content. These data suggest the existence of independent mechanism controlling the catabolism of the two amino acids.

Aminohydrolases↗
PubMed1978

Second-derivative spectroscopy of proteins. A method for the quantitative determination of aromatic amino acids in proteins.

Second derivative spectroscopy has been used to resolve the complex protein spectrum in the near-ultraviolet region and the contributions of the three aromatic chromophores have been evaluated. A method for the direct quantitative determination of phenylalanine and tryptophan in proteins has been carried out. Phenylalanine determination has been carried out in the spectral region between 250 and 265 nm, where there are no significant contributions from other aromatic chromophores. Tryptophan determination has been performed in the 290-295-nm region and the experimental values have been corrected for the presence of tyrosine. The results obtained on 10 highly purified proteins have been found in good agreement with those obtained from sequence analysis.

Phenylalanine↗
PubMed1978

Amino acid composition and physico-chemical properties of bluefin tuna (Thunnus thynnus) myoglobin.

1. The heart ventricle myoglobin of Atlantic bluefin tuna has been purified and its amino acid composition has been determined. 2. The perturbing effect of guanidine hydrochloride on the molecular structure of tuna ferrimyoglobin and its corresponding apoprotein has been investigated by Soret absorbance and ultraviolet fluorescence. 3. The conformation-free energy of unfolding delta G0 has been calculated by thermodynamic treatments of the data concerning guanidine unfolding. 4. The results have been compared with other known myoglobins, particularly those of yellowfin tuna.

Amino Acids↗
PubMed1978

Polyamine quantitative analysis in the developing chick embryo.

1. An improved specific and sensitive method for the separation and detection of polyamines, based on an automated ion exchange chromatography, is described. 4. The mean contents of polyamines in embryos at different stages of development are reported. The data show a direct correlation between the cellular polyamine content and the growth rate of the embryo.

Animals↗