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J Grassi

Publications and source records attributed to J Grassi.

119 records · Page 7Linked to original sources
PubMed1984

Synthesis in vitro of precursors of the catalytic subunits of acetylcholinesterase from Torpedo marmorata and Electrophorus electricus.

We translated poly(A-rich messenger RNA prepared from the electric organs of Electrophorus electricus and Torpedo marmorata in a reticulocyte lysate system. In the case of Electrophorus, which appears to contain only one type of acetylcholinesterase catalytic subunit, an anti-(Electrophorus acetylcholinesterase) antiserum precipitated a single 65-kDa polypeptide from the products translation obtained in vitro. In the case of Torpedo, where a number of distinct catalytic subunits corresponding to different fractions of the enzyme have been described, an anti-(Torpedo acetylcholinesterase) antiserum precipitated two main polypeptides, 61 kDa and 65 kDa, both of which could be displaced by unlabelled purified Torpedo acetylcholinesterase. Synthesis in vitro thus appears to produce a single type of precursor of the acetylcholinesterase catalytic subunit for Electrophorus, and at least two distinct precursors for Torpedo, suggesting that several mRNAs code for the catalytic subunits in the latter species.

Acetylcholinesterase↗
PubMed1984

An immunological study of rat acetylcholinesterase: comparison with acetylcholinesterases from other vertebrates.

We have examined the immunoreactivity of acetylcholinesterase from different vertebrate species with a rabbit antiserum raised against the purified rat brain hydrophobic enzyme (G4 form). We found no significant interaction with enzymes from Electrophorus, Torpedo, chicken, and rabbit. The antiserum reacted with acetylcholinesterases from the brains of the other mammalian species studied, with titers decreasing in the following order: rat = mouse greater than human greater than bovine. The serum was inhibitory with murine and human acetylcholinesterases, but not with the bovine enzyme. The inhibition was partially depressed in the presence of salt (e.g., 1 M NaCl). In those species whose acetylcholinesterase was recognized by the antiserum, both soluble and detergent-soluble fractions behaved in essentially the same manner, interacting with the same antibodies. The apparent immunoprecipitation titer was decreased in the presence of salt, and it did not make any difference whether NaCl was included in the solubilization procedure or added to the extracts. Both G1 and G4 forms of acetylcholinesterase in the soluble and detergent-soluble fractions were recognized by the antiserum, and in the case of the human enzyme, by monoclonal antibodies produced against human erythrocyte acetylcholinesterase. However, the monomer G1 showed a clear tendency to form smaller complexes and precipitate less readily than the tetramer G4. Although we cannot exclude the existence of significant differences between the various molecular forms of acetylcholinesterase, our results are consistent with the hypothesis that they all derive from the same gene or set of genes by posttranslational modifications.

Acetylcholinesterase↗
PubMed1983

Relationship of collagen-tailed acetylcholinesterase with basal lamina components. Absence of binding with laminin, fibronectin, and collagen types IV and V and lack of reactivity with different anti-collagen sera.

In view of their supposed localization in extracellular structures, such as basal lamina, we have investigated the possible interactions of collagen-tailed forms of acetylcholinesterase from Electrophorus and bovine superior cervical ganglion with matrix proteins: laminin, fibronectin and types IV and V collagens. Using binding and sedimentation assays, with iodinated or non-radioactive matrix proteins, we have not observed any significant interaction, in conditions of high or low ionic strength. We also examined whether the collagen tail of acetylcholinesterase asymmetric forms possessed an immunological relationship with known collagen types (I, III, IV, V) from mammalian sources. We found no specific immunoreactivity with any of the 32 sera studied, either with the iodinated Electrophorus or with the native bovine enzyme. We conclude from these negative results that the collagen-like tail of acetylcholinesterase is clearly distinct from the classical types of collagen and that asymmetric forms of the enzyme do not interact specifically with the matrix proteins studied. This does not exclude the possibility of specific interactions with other components, remaining to be identified.

Acetylcholinesterase↗
PubMedDate not supplied

Pharmacological modulation of interleukin 1 production by cultured endothelial cells from human umbilical veins.

Solid-phase enzyme immunoassays (with high-turnover acetylcholinesterase as label) for human IL-1 alpha and IL-1 beta were applied to quantify the production of these factors by cultured human umbilical vein endothelial cells (HUVECs). Immunoreactive IL-1s exhibited a typical pattern in HUVECs, under either basal or stimulated conditions: the alpha form was predominant over the beta form and the cell-associated IL-1s measured were more abundant than the material recovered in the supernatants. Bacterial lipopolysaccharide (LPS, 0.5-5 micrograms/ml) significantly increased the basal production of IL-1. Pulses of recombinant IL-1 alpha or -beta or of TNF-alpha followed by a 24 h culture period were also associated with an increased endothelial production of IL-1, with a higher proportion of material secreted in the supernatants as compared with LPS. Other cytokines applied as pulses failed to induce the IL-1s or to modify LPS-induced production of IL-1: they include IL-2, immune interferon, GM-CSF, TGF-beta and EGF. Pharmacological modulators of LPS-induced IL-1 production were identified: glucocorticoids were inhibitors whereas retinoic acid and 1.25-dihydroxy-vitamin D3 had no effects and prostaglandin E2 and IBMX were weak inhibitors. There is no evidence that IL-1 alpha and IL-1 beta are regulated differently in HUVECs, but several significant differences from the monocyte were observed in the regulation of HUVEC IL-1 production.

1-Methyl-3-isobutylxanthine↗
PubMedDate not supplied

Importance of hydropathic complementarity for the binding of the neuropeptide substance P to a monoclonal antibody: equilibrium and kinetic studies.

In a previous study (Boquet et al., 1995, Molec. Immunol. 32, 303-308) we observed remarkable inversions of hydropathic profiles between complementarity determining regions (CDRs) of an anti-substance P monoclonal antibody (SP31) and the corresponding 5 amino acid C-terminal peptide epitope. Here we demonstrate the importance this hydropathic complementarity by measuring the immunoreactivity of SP-related peptides which have been modified in their C-terminal parts so that hydropathic profile has been conserved (by substituting amino acids in the epitope) or modified (by mixing the sequence of amino acids in the epitope). Experiments performed in equilibrium conditions using a competitive enzyme immunoassay showed that most of the peptides conserving the hydropathic profile of SP epitope were recognized by mAb SP31 (even if marked variations in affinity were observed), while those for which the hydropathic profile was modified exhibited very low or undetectable affinity. The kinetic parameters (ka and kd) of peptide-antibody interactions were determined using Surface Plasmon Resonance technology (BIACORE 2000). These measurements showed that all peptides recognized by mAb SP31 had similar association rate constants (close to 2 x 10[5] M[-1] s[-1]), differences in binding affinities essentially resulting from differences in dissociation rate constants (ranging from 1.61 x 10[-4] to 1.15 x 10[-1] s[-1]). From these results, it was concluded that hydropathic complementarity between the epitope and the paratope could be a necessary but not sufficient condition for establishing high-affinity binding. We hypothesize that hydropathic interactions may play a critical role during the first contacts between antibody CDRs and the peptide, possibly by favouring reorganization of water molecules at the antibody-peptide interface.

Amino Acid Sequence↗
PubMedDate not supplied

Quantification of lamivudine (3TC) by competitive immunoassay.

Lamivudine or 3TC, the (-) eniantiomer of 2'-deoxy-3'-thiacytidine, is a prototype of a novel class of levogyre dideoxynucleosides analogues used in treatment of HIV and HBV infection. We describe a method corresponding to the first enzyme immunoassay for quantifying this antiviral drug. This technique use an enzyme conjugate that not require the use of radioactive labelling. In this study, anti-3TC antibodies were raised in rabbits by immunising with 3TC-HS-kelhoyle limpet hemocyanin (KLH) conjugate.

Anti-HIV Agents↗