STochastic Optical Reconstruction Microscopy (STORM) reveals the nanoscale organization of pathological aggregates in human brain.

AIMS: Histological analysis of brain tissue samples provides valuable information about the pathological processes leading to common neurodegenerative disorders. In this context, the development of novel high-resolution imaging approaches is a current challenge in neuroscience. METHODS: To this end, we used a recent super-resolution imaging technique called STochastic Optical Reconstruction Microscopy (STORM) to analyse human brain sections. We combined STORM cell imaging protocols with neuropathological techniques to image cryopreserved brain samples from control subjects and patients with neurodegenerative diseases. RESULTS: This approach allowed us to perform 2D-, 3D- and two-colour-STORM in neocortex, white matter and brainstem samples. STORM proved to be particularly effective at visualizing the organization of dense protein inclusions and we imaged with a <50 nm resolution pathological aggregates within the central nervous system of patients with Alzheimer's disease, Parkinson's disease, Lewy body dementia and fronto-temporal lobar degeneration. Aggregated Aß branches appeared reticulated and cross-linked in the extracellular matrix, with widths from 60 to 240 nm. Intraneuronal Tau and TDP-43 inclusions were denser, with a honeycomb pattern in the soma and a filamentous organization in the axons. Finally, STORM imaging of α-synuclein pathology revealed the internal organization of Lewy bodies that could not be observed by conventional fluorescence microscopy. CONCLUSIONS: STORM imaging of human brain samples opens further gates to a more comprehensive understanding of common neurological disorders. The convenience of this technique should open a straightforward extension of its application for super-resolution imaging of the human brain, with promising avenues to current challenges in neuroscience.

Antibody- and macrophage-mediated segmental demyelination in chronic inflammatory demyelinating polyneuropathy: clinical, electrophysiological, immunological and pathological correlates.

BACKGROUND AND PURPOSE: Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is a heterogeneous autoimmune disorder critically lacking diagnostic biomarkers. Autoantibodies to nodal and paranodal components have recently been described in a small subset of patients. Here, the diagnostic value of immune reactivity toward the myelin compartment was investigated. METHODS: Ninety-four French CIDP patients were retrospectively studied. The reactivity toward the peripheral nerve was investigated. Sural nerve biopsies were examined by electron microscopy and immunofluorescence. RESULTS: Twenty-one patients (22%) and three patients (3%) presented with a strong immunoglobulin G or immunoglobulin M reactivity respectively against the myelin compartment. The clinical, electrophysiological and morphological features were examined in nine of these patients for whom sural nerve biopsies were available. Seven patients were electrodiagnosed with definite CIDP, one with possible CIDP and one was unclassifiable but sural nerve biopsy argued for CIDP diagnosis. Electron microscopy of sural nerve biopsies demonstrated the presence of macrophage-mediated demyelination restricted to the internode in all nine patients. Immunolabelling for voltage-gated sodium channels, myelin and axonal markers confirmed the presence of segmental demyelination and of remyelination. The nodal and paranodal regions, however, were unaffected in these patients. Nerve conduction studies corroborated the multifocal and segmental profile, and seven patients showed increased duration of proximal (1.5-5.1 times) and/or distal (1.2-3.4 times) compound muscle action potential in at least two nerves. CONCLUSION: Antibody- and macrophage-mediated demyelination appears responsible for conduction alterations in CIDP patients and nerve immunostaining assays may serve as a supportive diagnostic biomarker.

Epidermal growth factor released from platelet-rich plasma promotes endothelial cell proliferation in vitro.

BACKGROUND AND OBJECTIVE: The therapeutic benefits of platelet-rich plasma (PRP) for the promotion of healing and regeneration of periodontal tissues are thought to result from enrichment in growth factors released from platelets. The aim of this study was to evaluate the effects of specific growth factors released from PRP on endothelial cell proliferation. MATERIAL AND METHODS: The levels of vascular endothelial growth factor (VEGF), platelet-derived growth factor BB (PDGF-BB), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) in supernatants of calcium- and thrombin-activated PRP samples from five donors were quantified by enzyme-linked immunosorbent assay. Supernatants were treated with neutralizing antibodies specific to each growth factor, and the effects of these treatments on human umbilical vein endothelial cell (HUVEC) proliferation in vitro were determined. The effect of removing EGF from PRP supernatants with antibody-coated beads on HUVEC proliferation was also tested. RESULTS: Average concentrations of VEGF, PDGF-BB, bFGF and EGF in PRP supernatants were 189, 27,190, 39.5 and 513 pg/mL, respectively. The addition of EGF neutralizing antibodies to the PRP supernatants significantly reduced HUVEC proliferation (up to 40%), while such an inhibition was not observed following neutralization of the other growth factors. Removal of EGF from PRP supernatants by treatment with antibody-coated beads also resulted in a significant decrease in HUVEC proliferation. Recombinant EGF increased HUVEC proliferation in vitro in a dose-dependent manner. CONCLUSION: This study showed that PRP supernatants are highly mitogenic for endothelial cells and provided evidence that this effect may be due, at least in part, to the presence of EGF. In vivo experiments are needed to confirm the roles of specific growth factors released from PRP in the healing of oral surgical and/or periodontal wounds.

Identification of the SH3 domain of GAP as an essential sequence for Ras-GAP-mediated signaling.

Guanosine triphosphatase activating protein (GAP) is an essential component of Ras signaling pathways. GAP functions in different cell types as a deactivator and a transmitter of cellular Ras signals. A domain (amino acids 275 to 351) encompassing the Src homology region 3 (SH3) of GAP was found to be essential for GAP signaling. A monoclonal antibody was used to block germinal vesicle breakdown (GVBD) induced by the oncogenic protein Ha-ras Lys12 in Xenopus oocytes. The monoclonal antibody, which was found to recognize the peptide containing amino acids 275 to 351 within the amino-terminal domain of GAP, did not modify the stimulation of the Ha-Ras-GTPase by GAP. Injection of peptides corresponding to amino acids 275 to 351 and 317 to 326 blocked GVBD induced by insulin or by Ha-Ras Lys12 but not that induced by progesterone. These findings confirm that GAP is an effector for Ras in Xenopus oocytes and that the SH3 domain is essential for signal transduction.

Cloning of a Grb2 isoform with apoptotic properties.

Growth factor receptor-bound protein 2 (Grb2) links tyrosine-phosphorylated proteins to a guanine nucleotide releasing factor of the son of sevenless (Sos) class by attaching to the former by its Src homology 2 (SH2) moiety and to the latter by its SH3 domains. An isoform of grb2 complementary DNA (cDNA) was cloned that has a deletion in the SH2 domain. The protein encoded by this cDNA, Grb3-3, did not bind to phosphorylated epidermal growth factor receptor (EGFR) but retained functional SH3 domains and inhibited EGF-induced transactivation of a Ras-responsive element. The messenger RNA encoding Grb3-3 was expressed in high amounts in the thymus of rats at an age when massive negative selection of thymocytes occurs. Microinjection of Grb3-3 into Swiss 3T3 fibroblasts induced apoptosis. These findings indicate that Grb3-3, by acting as a dominant negative protein over Grb2 and by suppressing proliferative signals, may trigger active programmed cell death.

A Ras-GTPase-activating protein SH3-domain-binding protein.

We report the purification of a Ras-GTPase-activating protein (GAP)-binding protein, G3BP, a ubiquitously expressed cytosolic 68-kDa protein that coimmunoprecipitates with GAP. G3BP physically associates with the SH3 domain of GAP, which previously had been shown to be essential for Ras signaling. The G3BP cDNA revealed that G3BP is a novel 466-amino-acid protein that shares several features with heterogeneous nuclear RNA-binding proteins, including ribonucleoprotein (RNP) motifs RNP1 and RNP2, an RG-rich domain, and acidic sequences. Recombinant G3BP binds effectively to the GAP SH3 domain G3BP coimmunoprecipitates with GAP only when cells are in a proliferating state, suggesting a recruitment of a GAP-G3BP complex when Ras is in its activated conformation.

Long-term hydroxytamoxifen treatment of an MCF-7-derived breast cancer cell line irreversibly inhibits the expression of estrogenic genes through chromatin remodeling.

Antiestrogen resistance is frequently observed in patients after longterm treatment with tamoxifen, a nonsteroidal antiestrogen widely used for endocrine therapy of breast cancer. In vitro studies in resistant cells showed that the expression of natural estrogen-responsive genes is frequently altered. Using MVLN cells, an MCF-7-derived cell model, we previously demonstrated that 4-hydroxytamoxifen (OHT) treatment irreversibly inactivated an estrogen-regulated chimeric luciferase response by a direct effect of the drug and not through a cell selection process (E. Badia et al., Cancer Res., 54: 5860-5866, 1994). In the present study, we present tamoxifen-resistant but still estrogen-dependent clones isolated after long-term treatment of MVLN cells with OHT and show that progesterone receptor (PR) expression was irreversibly decreased in some of these clones, whereas the PRA:PRB ratio of residual PR remained unchanged. The irreversible inactivation of both chimeric luciferase gene and PR gene expression was associated with the disappearance of DNase 1-hypersensitive sites. In the case of the chimeric gene, at least one of these sites was close to the estrogen responsive element. Genomic sequencing analysis of a clone with very low PR content did not reveal any methylation on CpG dinucleotides or any mutation in the PR gene promoter region. In all of the resistant clones tested and independently of their PR content, estrogen receptor expression was only lowered by half and remained functional, whereas pS2 expression was not modified. We also observed that the residual luciferase activity level (1-2%) of the MVLN clones, the luciferase expression of which had been irreversibly inactivated, was raised 4-fold by trichostatin A treatment. We conclude that long-term OHT treatment may modify the chromatin structure and thus could contribute to differentially silencing natural target genes.

Hydroxytamoxifen induces a rapid and irreversible inactivation of an estrogenic response in an MCF-7-derived cell line.

The MVLN cell line was established in our laboratory from MCF-7 cells by stable transfection with the luciferase gene under the control of an estrogen-responsive element from the Xenopus vitellogenin A2 gene. This cell line allowed us to visualize the induction by hydroxytamoxifen of a heterogeneity in the cell population with regard to the expression of the luciferase gene. Treated cells lost their estradiol-inducible luciferase activity, progressively and irreversibly; the luciferase expression of 80% of the cells was irreversibly inactivated by a 12-day hydroxytamoxifen treatment. We showed that this inactivation process was specific for an estrogenic response and was mediated by the estrogen receptor. Tamoxifen itself gave rise to such an inactivation, whereas other compounds belonging to the triphenylethylenic family but differently substituted on the ethylenic carbon and the ICI 164,384 compound were not as efficient. This irreversible inactivation was accompanied by a sharp decrease in the luciferase mRNA level; however, the estrogen receptor function and the cellular transcriptional machinery were not affected by the treatment. Although this antiestrogen treatment neither affected the estrogen-dependent cell growth nor irreversibly inhibited the expression of the natural pS2 gene, these results highly suggest that long-term antiestrogen therapy may lead to some heterogeneity in tumor cells throughout the course of patient treatment.

The Saccharomyces cerevisiae gene product SDC25 C-domain functions as an oncoprotein in NIH3T3 cells.

Ras proteins in mammalian cells cycle between a GTP-bound 'on' state and a GDP-bound 'off' state. Activation of Ras p21 results from the dissociation of tightly bound GDP and the exchange of bound GDP for GTP. A guanine nucleotide exchange factor is required for this activation. Activation promotes interaction with effector molecules and allows the signal to be transduced. In Saccharomyces cerevisiae, the function of guanine nucleotide exchange has been ascribed to the product of the CDC25 gene. The C-terminus domain of SDC25, a homologue of CDC25, can substitute for the CDC25 protein in yeast. We have demonstrated that the SDC25 C-terminus domain promotes GTP binding to Ras p21 in CHO cells. In the present study, we found that the stable expression of the SDC25 C-terminus domain induced transformation of NIH3T3 cells. Ras proteins in these tumorigenic cells were GTP bound. In addition, the coexpression of wild-type Ha-Ras protein with the SDC25 C-terminus was found to enhance the tumorigenic properties of the NIH3T3 cells. These results imply that, in subsets of human tumours, cellular Ras p21 might be found in its GTP-bound active form as a consequence of an oncogenic activation of a mammalian Ras guanine nucleotide exchange factor.

Phenylalanine ammonia-lyase in tobacco mosaic virus-infected hypersensitive tobacco. Density-labelling evidence of de novo synthesis.

A strong increase in phenylalanine ammonia-lyase (EC 4.3.1.5) activity occurs in tobacco mosaic virus-infected tobacco leaves developing necrotic local lesions. Comparison of physicochemical properties of the partially purified enzymes extracted from healthy and infected leaves showed that the hypersensitive reaction leads to an increase in the pool size of the same active enzyme molecules as those present in non-infected material. The molecular mechanism of enzyme formation was investigated by radiolabelling with [3H]leucine and by density labelling with 2H2O. Abnormal patterns of incorporation of radioactivity into all soluble proteins were found in infected leaves carrying local lesions. In contrast, uptake of deuterium into the amino acid pool was the same in healthy and infected leaves. Unstimulated phenylalanine ammonia-lyase was shown to be a long-lived enzyme (half-life: 25-35 h). Results of comparative density labelling experiments unequivocally demonstrated that the increased enzyme pool size arose from an increased rate of synthesis mediated by the hypersensitive reaction.

Estrogenic activity of cosmetic components in reporter cell lines: parabens, UV screens, and musks.

In this work, the estrogenic effects of three classes of substances included in cosmetic formulations-parabens, ultraviolet (UV) screens, and musk fragrances-were studied. Their estrogenic activity was measured with the use of three reporter cell lines: HELN, HELN ERalpha, and HELN ERbeta. These three cell lines allowed for the measurement of estrogenic activity toward estrogen receptors alpha and beta (ERalpha and ERbeta, while taking nonspecific interactions into account. Eight of the 15 substances tested showed specific estrogenic activity with the following degree of potency on ERalpha butylparaben > propylparaben > homosalate = octyl-dimethyl-PABA = 4-methyl-benzylidenecamphor = octyl-methoxycinnamate > ethylparaben = galaxolide. Among these active substances, parabens activated ERalpha and ERbeta similarly, UV screens activated ERalpha moderately and had almost no effect on ERbeta, and fragrances did not activate ERbeta. Methylparaben, ethylparaben, musk moskene, celestolide, and cashmeran did not activate estrogenic responses up to 10(-5) M. Musk ketone and benzophenone-3 were not considered estrogenic at 10(-5) M.

Intracellular expression and functional properties of an anti-p21Ras scFv derived from a rat hybridoma containing specific lambda and irrelevant kappa light chains.

A rat single-chain Fv (Y238 scFv) was derived from the Y13-238 monoclonal antibody, a non-neutralizing anti-Ras antibody. The Y13-238 hybridoma expresses two functional light chains. N-terminus microsequencing of these chains showed the presence of the Y3 Ag1.2.3 Vkappa chain derived from the rat fusion partner and of a rat Vlambda chain. Primers designed for rat Vlambda amplification allowed the cloning of a functional scFv that could bind p21Ras. The kinetics of interaction of purified Y238 scFv with the p21Ras protein was evaluated by BIAcore with a NTA sensor chip and gave an apparent affinity constant in the nanomolar range (K(D)=4.58+/-0.63 nM). Immunoprecipitation experiments of Y238 scFv expressed in Xenopus laevis oocytes confirmed the specificity of the scFv for the Ras protein. Y238 scFv could be intracellularly expressed in oocytes and in mammaliam cells without adverse effect on the Ras signalling cascade. This scFv was therefore used as control in experiments where another anti-Ras scFv (Y259 scFv, derived from the neutralizing anti-Ras mAb Y13-259) blocked the Ras pathway in vitro and led to tumor regression in a nude mouse model [Cochet, O., Kenigsberg, M., Delumeau, I., Virone-Oddos, A., Multon, M.C., Fridman, W.H., Schweighoffer, F., Teillaud, J.L., Tocqué, B., 1998. Intracellular expression of an antibody fragment-neutralizing p21 ras promotes tumor regression. Cancer Res. 58, 1170-1176.]. Finally, BIAcore analyses indicated that the epitopes recognized by Y238 and Y259 scFvs are not overlapping and allowed a more precise definition of the Y13-238 epitope.

Estrogen regulation in human breast cancer cells of new downstream gene targets involved in estrogen metabolism, cell proliferation and cell transformation.

We explored, by cDNA mini-arrays, gene expression measurements of MVLN, a human breast carcinoma cell line derived from MCF-7, after 4 days of exposure to 17beta-estradiol (E(2)) treatment, in order to extend our understanding of the mechanism of the pharmacological action of estrogens. We focused on 22 genes involved in estrogen metabolism, cell proliferation regulation and cell transformation. The specificity of the E(2) response was reinforced by comparison with 4-hydroxytamoxifen (OH-Tam), ICI 182,780 and E(2)+OH-Tam expression profiles. Real-time quantitative PCR (RTQ-PCR) confirmed the variation of expression of known (TFF1, AREG, IRS1, IGFBP4, PCNA, ERBB2, CTSD, MYC) as well as novel (DLEU2, CCNA2, UGT1A1, ABCC3, ABCC5, TACC1, EFNA1, NOV, CSTA, MMP15, ZNF217) genes. The temporal response of these gene expression regulations was then investigated after 6 and 18 h of E(2) treatment and this allowed the identification of different time-course patterns. Cycloheximide treatment studies indicated first that estrogen affected the transcript levels of ABCC3 and ABCC5 through dissimilar pathways, and secondly that protein synthesis was needed for modulation of the expression of the CCNA2 and TACC1 genes by estrogens. Western blot analysis performed on TFF1, IRS1, IGFBP4, amphiregulin, PCNA, cyclin A2, TACC1 and ABCC5 proteins confirmed the mini-array and RTQ-PCR data, even for genes harboring low variations of mRNA expression. Our findings should enhance the understanding of changes induced by E(2) on the transcriptional program of human E(2)-responsive cells and permit the identification of new potential diagnostic/prognostic tools for the monitoring of estrogen-related disease conditions such as breast cancer.

Steroidal affinity labels of the estrogen receptor. 3. Estradiol 11 beta-n-alkyl derivatives bearing a terminal electrophilic group: antiestrogenic and cytotoxic properties.

With the aim of developing a new series of steroidal affinity labels of the estrogen receptor, six electrophilic 11 beta-ethyl (C2), 11 beta-butyl (C4), or 11 beta-decyl (C10) derivatives of estradiol bearing an 11 beta-terminal electrophilic functionality, i.e. bromine (C4), (methylsulfonyl)oxy (C2 and C4), bromoacetamido (C2 and C4), and (p-tolylsulfonyl)oxy (C10), were synthesized. The range of their affinity constants for binding the estrogen receptor was 0.4-37% that of estradiol; the order of increasing affinity (i) relative to the 11 beta-alkyl arm was ethyl < butyl and (ii) relative to the electrophilic functionality was bromoacetamido < bromine < (methylsulfonyl)oxy. Regardless of the conditions used, including prolonged exposure of the receptor to various pH levels (7-9) and temperatures (0-25 degrees C), the extent of receptor affinity labeling by the 11 beta-ethyl and 11 beta-butyl compounds, if any, was under 10%. This was in sharp contrast to results obtained using 11 beta-((tosyloxy)decyl)estradiol which labeled from 60% to 90% of the receptor hormone-binding sites with an EC50 of approximately 10 nM. Estrogenic and antiestrogenic activities of the compounds were determined using the MVLN cell line, which was established from the estrogen-responsive mammary tumor MCF-7 cells by stable transfection of a recombinant estrogen-responsive luciferase gene. The two 11 beta-ethyl compounds were mainly estrogenic, whereas the three 11 beta-butyl and the 11 beta-decyl compounds essentially showed antiestrogenic activity. The fact that the chemical reactivities of 11 beta-ethyl and 11 beta-butyl compounds were not compromised by interaction with the estrogen receptor made the synthesized high-affinity compounds potential cytotoxic agents which might be able to exert either (i) a specific action on estrogen-regulated genes or (ii) a more general action in estrogen-target cells. Therefore the ability of the compounds (1) to irreversibly abolish estrogen-dependent expression of the luciferase gene and (2) to affect the proliferation of MVLN cells were determined. All electrophiles were able to irreversibly suppress expression of the luciferase gene; the antiestrogenic electrophiles were more potent than the estrogenic ones but less efficient than 4-hydroxytamoxifen, a classical and chemically inert triphenylethylene antiestrogen. Only the antiestrogenic electrophiles decreased cell proliferation; however, they were less potent than 4-hydroxytamoxifen. In conclusion, the synthesized electrophilic estradiol 11 beta-ethyl and 11 beta-butyl derivatives (i) were not efficient affinity labels of the estrogen receptor and (ii) did not display significant cytotoxicity in estrogen-sensitive mammary tumor cells. However, since these derivatives displayed high affinity for the estrogen receptor, they could be used to prepare potential cytotoxic agents which might be selective for tumors affecting estrogen-target tissues, by coupling them with a toxic moiety.

Inhibition of platelet aggregation by human placenta.

Human placental cytosol inhibits platelet aggregation induced by high doses of collagen. The aim of this study was to investigate whether this anti-aggregating activity was caused only by the presence of various activities already described in the placenta (an ADP-consuming enzyme, a fatty acid cyclooxygenase inhibitor, and a thromboxane synthetase inhibitor) or whether another factor was present. Heating the cytosol at 50 degrees C for 6 min destroyed the inhibitor of collagen-induced aggregation. ADPase and the AA pathway inhibitors were not modified by this treatment. We therefore show the presence of an additional anti-aggregating factor: it is destroyed by heating at 50 degrees C. We also tested for the presence of an inhibitor of AA release in the placental cytosol using three different methods (rabbit platelets in PRP, washed rabbit platelets, and NRK fibroblasts) but no inhibition could be evidenced. We conclude that this new anti-aggregating factor, which is probably a protein, acts neither through AA release inhibition nor AA cascade inhibition.

MVLN cells: a bioluminescent MCE-7-derived cell line to study the modulation of estrogenic activity.

We previously established a stable expression system in MCF-7 cells for the detection of (anti)estrogenic activity by assaying the reporter enzyme activity of firefly luciferase. In this cell line (called MVLN), the bioluminescent response can be measured either in the cellular homogenate, or in intact living cells. Here we present various potential experimental uses of this cellular model. First, we used this cell line to screen natural or synthetic molecules classified as full or partial (anti)estrogens and observed that their behavior towards our model was identical to that expected. Moreover, the bioluminescent response was in agreement with the natural responses like cellular proliferation or stimulation of the progesterone receptor. We then demonstrated the inhibitory effects of retinoic acid and 1,25 dihydroxyvitamin D3, two molecules which do not compete with estradiol for its receptor. We thus deduced that with this cell line an "antiestrogenic" effect which occurred at any step of the estrogenic action, might be detected. Finally, we showed that detection of luciferase activity in intact living cells was particularly helpful for investigating the evolution of estrogenic activity. For instance, we observed that long-term treatment of MVLN cells with an antiestrogen irreversibly decreased the bioluminescent response by more than 90%. This phenomenon affected all cells equally and could not be reversed, even by long-term estradiol treatment. We therefore conclude that this chimeric response faithfully reflects estrogenic action in the cell and can be used to develop different aspects of the endocrine research.

Identification and tissue localization of an eosinophil 17 kDa protein accumulating in rat uterus upon estradiol treatment.

In a previous paper (J. Steroid Biochem. 29 (1988) 475-480), the isolation of a 17 kDa protein that was dramatically induced in the uterus of estrogen-treated spayed rats was presented. We now describe a new purification procedure that is compatible with microsequencing of the 17 kDa protein. The protein partial N-terminal amino acid sequence analysis gave 28 residues that revealed a strong homology to the human major basic protein (MBP) of eosinophils described by Wasmoen et al. (J. Biol. Chem. 263 (1988) 12559-12563). Polyclonal rabbit antibodies were raised against this protein and used for tissue or blood cell analysis after electrophoresis and Western blotting. The 17 kDa protein was found to be constitutively present in the stomach and small intestine of the rat and guinea-pig. Estrogen treatment had a clearcut effect in guinea-pig uterus, but not as drastic as that observed in rat uterus. The protein was abundant in purified rat eosinophils. The antibodies cross-reacted with human MBP and an equivalent molecular weight human polymorphonuclear leukocyte protein. Immunohistochemical staining of rat uterus sections showed that the protein was first only associated with eosinophils that emigrate upon estrogen treatment; it then spread throughout the stroma and the deep glandular epithelium. It was not found in the myometrium. In conclusion, the appearance of a 17 kDa protein that is presumably the rat MBP is clearly regulated in the rat uterus.

Polypeptide composition of an adenovirus type 5 used in cancer gene therapy.

For cancer gene therapy, a recombinant adenovirus serotype 5 named RPR/INGN201 has been constructed by susbtitution of the E1 region with human tumor suppressor gene p53. The protein components of RPR/INGN201 virions were separated by reversed-phase HPLC and were individually identified by electrospray time-of-flight mass spectrometry and N-terminal sequencing, both on intact proteins and on their proteolytic fragments after trypsin digestion. Twenty-five peptide components of the proteome (including fiber) with greater than 0.25-0.5% contribution to the protein content of the virus were identified and characterized. Fiber was confirmed to be partially glycosylated (both the non-glycosylated and the monoglycosylated states were identified), and two proteins were isolated and identified as phosphorylation derivatives, namely protein V (non-phosphorylated and monophosphorylated) and protein IIIa (mono- and diphosphorylated). This new analytical tool proved to be very useful not only for refining our current knowledge of the polypeptide repertoire of purified infectious virions but also for monitoring and very rapidly identifying structural modifications resulting from changes in the manufacturing process. It was also used successfully for the characterization of various adenoviral constructs.

The up-regulation of vascular endothelial growth factor in mutated Ha-ras HaCaT cell lines is reduced by a farnesyl transferase inhibitor.

The induction of tumor angiogenesis is mediated in particular by an increased production of VEGF. As ras oncogene is implicated in tumorigenesis, the inhibition of farnesyl transferase activity has recently been developed. The purpose of this study was to evaluate whether expression of mutated Ha-ras oncogene is associated with an altered expression of VEGF in an in vitro model of human skin carcinogenesis and to appreciate the effect of a new farnesyl transferase inhibitor on this VEGF expression. The amounts of VEGF secreted by an HaCaT cell line and two cell clones (metastatic or not) obtained after mutated c-Ha-ras transfection were compared. Our findings showed that the release of VEGF is greater for HaCaT-ras than for HaCaT cells and could be down-regulated using a protein farnesyl transferase inhibitor, in a reversible and dose-dependent manner. These results confirm that the Ha-ras oncogene can contribute to tumor development and progression of epidermal tumors through neoangiogenesis and that farnesyl transferase inhibitors as anticancer drugs may be efficient for the reduction of skin tumor growth.

[Complement markers Bf and C4 in multiple sclerosis]. / Les marqueurs Bf et C4 du complément dans la sclérose en plaques.

Some immunogenetic HLA markers are significantly correlated with multiple sclerosis, e.g.: the antigens B7, DR2, and the associations B7-DR2, A3-B7-DR2. In addition, the polymorphism of the allotypes Bf and C4 is also controlled by chromosome 6; a study of these markers is therefore of interest. The study of Bf and C4 in multiple sclerosis included a population of genotyped unrelated patients: 50 patients for Bf markers and 41 for C4A and C4B markers. This study revealed an over-representation of allotype S and homozygous BfSS in multiple sclerosis. BfSS homozygote was significantly more frequent in the B7 negative and/or DR2 negative patients, i.e. when the risk markers per se were absent. No correlation could be evidenced with the remittent or progressive character of the disease. These data, obtained from the study of C4, are still preliminary ones. The results found with the Bf markers confirm the existence of a genetic factor in multiple sclerosis and suggest that the susceptibility gene of the disease could be closer to locus Bf than to locus DR.