Serge Chwetzoff

A monoclonal antibody recognizing a conserved epitope in a group of phospholipases A2

Notexin and nigexine are monomeric phospholipases A2(PLA2s) from the venoms of Notechis scutatus scutatus and Naja nigricollis, respectively. Polyclonal antibodies raised in mice against these antigenic proteins displayed non-reciprocal cross-reactivity; anti-notexin antibodies recognized notexin but not nigexine, whereas anti-nigexine antibodies recognized both antigens. Polyclonal antibodies raised by successive immunization with nigexine and notexin contained cross-reacting antibodies with affinities for both antigens that differed from those of antibodies present in anti-nigexine antiserum. A monoclonal antibody has been obtained from a mouse immunized with both PLA2s. This monoclonal antibody, called MN1, recognized notexin and nigexine with comparable high affinity (Kd = 10(-9) M). It also recognized most purified PLA2s from elapid snake venoms and all PLA2-containing venoms from cobras and sea-snakes. This offers the first demonstration that most PLA2s from cobras and sea-snakes share a fine structure which is not restricted to the common catalytic site.

Immunological properties of notexin, a potent presynaptic and myotoxic component from venom of the Australian tiger snake Notechis scutatus scutatus

Neutralizing antibodies were raised in mice against notexin, the most toxic phospholipase A2 (PLA2) from Notechis scutatus scutatus venom, without the necessity of detoxifying the toxin prior to immunization. Using a sensitive radioimmunoassay we demonstrated that anti‐notexin antibodies recognized (i) the parent antigen, (ii) closely related isoforms of notexin and (iii) venoms from Notechis genus snakes. In contrast, they failed to recognize other purified PLA2 or PLA2‐containing venoms from other origins. Substitutions or chemical modifications occurring in the C‐terminal part of the polypeptide chain of notexin altered the binding affinity for antibodies, implying that this region constitutes an antigenic domain of notexin.

On the purification of notexin: Isolation of a single amino acid variant from the venom of Notechis scutatus scutatus

Venom of the Australian tiger snake, Notechis scutatus scutatus was fractionated by conventional ion‐exchange chromatography. The fraction containing notexin, a well‐known single‐chain toxic phospholipase A2, was further purified by reverse‐phase high‐performance liquid chromatography. Two main components were isolated and the major one corresponded to notexin. The other component, designated as notechis Ns, was an isofonn of notexin. Notechis Ns and notexin possessed similar in vitro esterase activity, in vitro neuromuscular activity and in vivo lethality. Amino acid composition and sequence of the Staphylococcus aureus V8‐protease peptides demonstrated that primary structures of notechis Ns and notexin differed from each other by a single substitution amongst 119 amino acids: Lys → Arg at position 16.

Ubiquitin is physiologically induced by interferons in luminal epithelium of porcine uterine endometrium in early pregnancy: global RT-PCR cDNA in place of RNA for differential display screening1

Early in the course of pregnancy, at the preimplantation stage, the pig embryo is likely to exert a paracrine effect on the tissue intended to receive it, via the secretion of interferons. Our observations show that trophoblastic interferons induce an increase of some mRNAs in the epithelial cells of the gilt endometrium, which would illustrate this phenomenon. The increase of four mRNAs, whose corresponding cDNAs are dD1, dD2, dD3 and dD4, has been examined in this study. The method used is similar to Northern blot analysis except that mRNAs in the blot are replaced by cDNAs produced from total cellular poly(A)+ mRNAs by global everse‐ ranscription olymerase hain eaction (RT‐PCR). Northern blot hybridization requires a considerable quantity of starting material – which we estimate in this study to be several million porcine endometrium cells – whereas the RT‐PCR‐based method gives comparable results starting with only a few cells – about 200. Using this method, the differential nature of dD1, dD2, dD3 and dD4 was shown. dD2 and dD3 correspond to genes already identified as interferon‐induced: the β2‐microglobulin and inkel‐Biskis‐Reilly murine sarcoma virus‐ ssociated biquitously secreted protein (FAU). dD1 corresponds to a still unidentified gene. dD4 encodes for the porcine UbA52 ubiquitin. Up to now, the increase in ubiquitin mRNA as a result of interferon effect has not been reported and is discussed in view of recent publications.

Evidence that the anti-coagulant and lethal properties of a basic phospholipase A2 from snake venom are unrelated

The basic phospholipase A2 (PLA2) from venom of the African elapid Naja nigricollis was previously shown to have anti‐coagulant and lethal properties, both of which were abolished by treatment with p‐bromophenacyl bromide (pBP). In the present paper we first report that pBP‐treated PLA2 is capable of inhibiting the anti‐coagulant activity but not the lethal activity of native PLA2, thus suggesting that both properties might be independent. We then confirm this evidence using PLA2‐specific monoclonal immunoglobulins. One of these, called HSF, neutralized the lethal activity but not the anti‐coagulant activity, whereas another antibody, called HSP2, inhibited the anti‐coagulant activity but not the lethal activity of the PLA2. The data presented in this paper are taken as evidence that the anti‐coagulant activity is not implicated in the lethal effects of basic PLA2 from Naja nigricollis.