p53 directly transactivates Δ133p53α, regulating cell fate outcome in response to DNA damage.

We have previously reported that the human p53 gene encodes at least nine different p53 isoforms, including Δ133p53α, which can modulate p53 transcriptional activity and apoptosis. In this study, we aimed to investigate the regulation of Δ133p53α isoform expression and its physiological role in modulating cell cycle arrest and apoptosis. We report here that in response to a low dose of doxorubicin (which induces cell cycle arrest without promoting apoptosis), p53 directly transactivates the human p53 internal promoter, inducing Δ133p53α protein expression. The induced Δ133p53α then inhibits p53-dependent apoptosis and G1 arrest without inhibiting p53-dependent G2 arrest. Therefore, endogenous Δ133p53α does not exclusively function in a dominant-negative manner toward p53, but differentially regulates cell cycle arrest and apoptosis.

Use of the tape stripping technique for directly quantifying esterase activities in human stratum corneum.

The enzymes secreted in the intercellular spaces of stratum corneum (SC), the outermost layer of the epidermis, are thought to be involved in normal desquamation and skin barrier function. Their activity can barely be measured due to the difficulty in isolating enough biological material. Human SC layers were obtained from the forearm of healthy volunteers by the tape stripping technique. Assays for esterase activities were carried out in specially designed plates which contained the SC blotted on tape strips, using various fluorescent methylumbelliferone acyl esters as substrates. Triacylglycerol hydrolase activities were also studied by this method. By using radiolabeled triolein and fluorescent 4-methylumbelliferyl 7-oleate as substrates, true lipase activities could be detected and quantitated in SC at pH 5.5 and 7.5. These activities were shown to be strongly inhibited by tetrahydrolipstatin while this was not the case with 4-methylumbelliferyl 7-heptanoate. The method described here combines the painless tape stripping technique with a sensitive plate assay analysis. Since the whole process needs little manipulation, this method can permit rapid quantitation of multiple enzyme activities from a single strip. Therefore, it will permit the study of the involvement of enzyme activities in epidermis aging and skin pathologies.

The V domain of herpesvirus Ig-like receptor (HIgR) contains a major functional region in herpes simplex virus-1 entry into cells and interacts physically with the viral glycoprotein D.

The herpesvirus entry mediator C (HveC), previously known as poliovirus receptor-related protein 1 (PRR1), and the herpesvirus Ig-like receptor (HIgR) are the bona fide receptors employed by herpes simplex virus-1 and -2 (HSV-1 and -2) for entry into the human cell lines most frequently used in HSV studies. They share an identical ectodomain made of one V and two C2 domains and differ in transmembrane and cytoplasmic regions. Expression of their mRNA in the human nervous system suggests possible usage of these receptors in humans in the path of neuron infection by HSV. Glycoprotein D (gD) is the virion component that mediates HSV-1 entry into cells by interaction with cellular receptors. We report on the identification of the V domain of HIgR/PRR1 as a major functional region in HSV-1 entry by several approaches. First, the epitope recognized by mAb R1. 302 to HIgR/PRR1, capable of inhibiting infection, was mapped to the V domain. Second, a soluble form of HIgR/PRR1 consisting of the single V domain competed with cell-bound full-length receptor and blocked virion infectivity. Third, the V domain was sufficient to mediate HSV entry, as an engineered form of PRR1 in which the two C2 domains were deleted and the V domain was retained and fused to its transmembrane and cytoplasmic regions was still able to confer susceptibility, although at reduced efficiency relative to full-length receptor. Consistently, transfer of the V domain of HIgR/PRR1 to a functionally inactive structural homologue generated a chimeric receptor with virus-entry activity. Finally, the single V domain was sufficient for in vitro physical interaction with gD. The in vitro binding was specific as it was competed both by antibodies to the receptor and by a mAb to gD with potent neutralizing activity for HSV-1 infectivity.

The human poliovirus receptor related 2 protein is a new hematopoietic/endothelial homophilic adhesion molecule.

We have recently described Poliovirus Receptor Related 2 (PRR2), a new cell surface molecule homologous to the poliovirus receptor (PVR/CD155). Both molecules are transmembrane glycoproteins belonging to the Ig superfamily (IgSF). They contain 3 Ig domains of V, C2, and C2 types in their extracellular regions that share 51% aa identity. The PRR2 gene encodes two mRNA isoforms of 3.0 kb (hPRR2 [short form]) and 4.4 kb (hPRR2delta [long form]), both widely expressed in human tissues, including hematopoietic cells. To further characterize PRR2 expression during hematopoiesis and to analyze its function, we have developed a monoclonal antibody (MoAb) directed against its extracellular region (R2.477). PRR2 was expressed in 96% of the CD34(+), 88% of the CD33(+), and 95% of the CD14(+) hematopoietic lineages and faintly in the CD41 compartment. Ectopic expression of both PRR2 cDNAs induced marked cell aggregation. A soluble chimeric receptor construct with the Fc fragment of human IgG1 (PRR2-Fc) as well as a fab fragment of the anti-PRR2 MoAb (R2.477) inhibit aggregation. PRR2-Fc binds specifically to PRR2-expressing cells. These results suggest that PRR2 is a homophilic adhesion receptor. PRR2 was also expressed at the surface of endothelial cells at the intercellular junctions of adjacent cells but not at the free cellular edges. Homophilic interactions are associated with dimerization of isoforms of PRR2 and lead to the tyrosine phosphorylation of PRR2delta. Altogether, these results suggest that homophilic properties of PRR2 could participate to the regulation of hematopoietic/endothelial cell functions.

Interfacial binding of human gastric lipase to lipid monolayers, measured with an ELISA.

Two sandwich enzyme linked immunosorbent assays (ELISA) were developed for evaluating the surface excess at the lipid/water interface of the human gastric lipase (HGL) and two anti-HGL monoclonal antibodies (mAbs). These assays were adapted to the monomolecular film technique used previously for measuring lipase kinetics. HGL and the two anti-HGL mAbs (4-3 and 218-13) were biotinylated without any significant loss of their biological activities occurring. They were further detected by ELISA using either anti-HGL or anti-mouse IgG polyclonal antibodies as specific captors before being revealed using a streptavidin--peroxidase conjugate as tracer. The detection limit was 25 and 85 pg in the case of HGL and mAb, respectively. By combining the above sandwich ELISA technique with the monomolecular film technique, it was possible for the first time to measure the enzymatic activity of HGL on 1,2-didecanoyl-sn-glycerol (dicaprin) monolayers as well as to determine the corresponding interfacial excess of the enzyme. The HGL turnover number increased steadily with the lipid packing. The specific activities determined on dicaprin films spread at 35 mN.m-1 were found to be in the range of the values measured under optimal bulk assay conditions, using tributyrin emulsion as a substrate [i.e., 1000 mumol/(min.mg of enzyme)]. At a given lipase concentration in the water subphase, the interfacial binding of HGL to the nonhydrolyzable egg yolk phosphatidylcholine (egg PC) monolayers was found to be 10 times lower than that in the case of dicaprin monolayers.(ABSTRACT TRUNCATED AT 250 WORDS)

Human pancreatic lipase. Importance of the hinge region between the two domains, as revealed by monoclonal antibodies.

Several monoclonal antibodies (mAbs) were prepared against human pancreatic lipase (HPL). Two enzyme-linked immunosorbent assay (ELISA) procedures were set up for screening hybridomas producing specific antibodies. Four mAbs (81-23, 146-40, 315-25, and 320-24) of the IgG1 isotype were found to react with HPL in both simple sandwich and double sandwich ELISAs, while mAb 248-31, of the IgG2b isotype, reacted only with HPL in a double sandwich ELISA. The results of Western blot analysis carried out with native and SDS-denatured HPLs indicated that mAb 248-31 recognized only native HPL, while all the other mAbs recognized both forms of HPL. Since mAb 248-31 did not recognize SDS-denatured HPL, it was not possible to localize its epitope. To carry out epitope mapping along the primary sequence of HPL, four fragments (14, 26, 30, and 36 kDa) resulting from a limited chymotryptic cleavage of HPL were characterized by Western blotting as well as N-terminal amino acid sequence analysis. Of the above five anti-HPL mAbs, four (81-23, 248-31, 315-25, and 320-24) were found to inhibit the lipolytic activity of HPL (in both the presence and absence of bile salts and colipase), while mAb 146-40 had no inhibitory effects. The epitope recognized by mAb 146-40 was found to be located in the N-terminal domain (Lys1-Phe335). Combined immunoinactivation and epitope mapping studies showed that three inhibitory mAbs (81-23, 315-25, and 320-24) recognize overlapping epitopes from the hinge region between the N- and C-terminal domains of HPL, belonging to the 26-kDa fragment. In the presence of lipids, a significant decrease has been observed in the bending angle between the N- and C-terminal domains of the HPL tertiary structure (van Tilbeurgh, H., Egloff, M. P., Martinez, C., Rugani, N., Verger, R. and Cambillau, C. (1993) Nature 362, 814-820). From the present immunochemical data, we further propose that locking the hinge movement with mAbs may induce lipase immunoinactivation.

Tryptic cleavage of gastric lipases: location of the single disulfide bridge.

Human (HGL) and rabbit (RGL) gastric lipases were cleaved by trypsin and the resulting peptides were characterized. Exposure of HGL to trypsin led to the production of three identified fragments (H1, H2 and H3) resulting from cleavage sites at Lys-4 and Arg-229. Fragments H2 (Lys-4-Arg-229) and H3 (Glu-230-Lys-379) were derived from fragment H1 (Lys-4-Lys-379). The single disulfide bridge (Cys-236-Cys-244) of the molecule is localized in fragment H3. Out of the three cysteine residues conserved in all known gastric lipases, the free sulfhydryl group (Cys-227) was localized in fragment H2. Immunoblots, carried out with the tryptic fragments of HGL and anti-HGL mAbs, revealed that five inhibitory mAbs immunoreacted selectively with the N-terminal fragment H2, whereas two other non inhibitory mAbs immunoreacted exclusively with the C-terminal fragment H3. Trypsin also cleaved RGL at two sites (Arg-55 and Arg-229) leading to four identifiable fragments (R1, R2, R3 and R4). One cleavage site (Arg-229) was found to be identical in both RGL and HGL. We propose that this latter site is localized between the two domains of native gastric lipases.