Stage- and tissue-specific expression of a beta-1,4-galactosyltransferase in the embryonic epidermis.

Changes in oligosaccharide structures of glycoconjugates have been observed, and are postulated to have key roles in embryonic development and differentiation. N-Acetylglucosamine (GlcNAc) beta-1,4-galactosyltransferase (beta4GalT) AKI showed different expression patterns in time and space, and different enzymatic activity from the other known family members. The epidermis of mouse embryo included a high level of AKI activities, which transferred galactose (Gal) to endogenous glycoprotein (molecular weight 130 kDa) (GP130). The maximum activity was for 13.5-d postcoitum embryos. Specific antibody against AKI inhibited 81% of GlcNAc betaGalT activities, which indicates that AKI represents the major part of the embryonic epidermis enzymes. AKI shows 2.2 times higher galactosyltransferase activity toward Gal-acceptor glucose with alpha-lactalbumin (alpha-LA) than toward GlcNAc without alpha-LA. AKI is also expressed in mouse melanoma and leukemia cell lines and in human basal cell carcinoma specimens. The GP130 Gal acceptor once galactosylated by AKI may be directly involved in epidermal differentiation and oncogenesis.

Dorsal dermis development depends on a signal from the dorsal neural tube, which can be substituted by Wnt-1.

To investigate the origin and nature of the signals responsible for specification of the dermatomal lineage, excised axial organs in 2-day-old chick embryos were replaced by grafts of the dorsal neural tube, or the ventral neural tube plus the notochord, or aggregates of cells engineered to produce Sonic hedgehog (Shh), Noggin, BMP-2, Wnt-1, or Wnt-3a. By E10, grafts of the ventral neural tube plus notochord or of cells producing Shh led to differentiation of cartilage and muscles, and an impaired dermis derived from already segmented somites. In contrast, grafts of the dorsal neural tube, or of cells producing Wnt-1, triggered the formation of a feather-inducing dermis. These results show that the dermatome inducer is produced by the dorsal neural tube. The signal can be Wnt-1 itself, or can be mediated, or at least mimicked by Wnt-1.

Localisation of members of the notch system and the differentiation of vibrissa hair follicles: receptors, ligands, and fringe modulators.

Hair vibrissa follicle morphogenesis involves several cell segregation phases, in the dermis as well as in the epidermis. The expression of Notch-related genes, which are well established mediators of multiple cell segregation events in Drosophila development, was studied by in situ hybridisation during embryonic mouse vibrissa follicle morphogenesis and the first adult hair cycle. The results show that two receptors, Notch1 and -2, three ligands, Delta1, Serrate1, and -2, and the three Fringe regulators, Lunatic, Manic, and Radical, are expressed in different locations and morphogenetic stages. First, the appearance of hair vibrissa primordia involves the expression of complementary patterns of Notch2, Delta1, and Lunatic Fringe in the dermis and of Notch1, Serrate2, and Lunatic Fringe in the epidermis. Second, this expression pattern is no longer found after stage 3 in the dermis. Meanwhile, in the epidermis, the expression of Notch1, Serrate2, and Lunatic Fringe before the formation of the placode may be involved in determining two populations of epidermal cells in the developing follicle. Third, complementary expression patterns for Notch1, Manic, and Lunatic Fringe, as well as Serrate1 and -2 as previously shown (Powell et al., 1998), are progressively established from stage 4 of embryonic development both in the outer root sheath and in the hair matrix. These patterns are consistent with the one found in the adult anagen phase. During the hair vibrissa cycle, Notch1 and Manic Fringe display temporal and spatial changes of expression, suggesting that they may intervene as modulators of trichocyte activities.

Differential expression pattern of the three Fringe genes is associated with epidermal differentiation.

Epidermal differentiation, as keratinocytes go through different layers to the skin surface, may imply a differential activation of Notch transmembrane proteins. In mouse, as recently shown in Drosophila, Notch activation by its ligands may be modulated by Fringe secreted proteins. Therefore, we cloned the mouse homolog of Radical-fng, synthesized riboprobes for Lunatic-fng, Manic-fng, and Radical-fng, and examined their expression during epidermal differentiation. Expression of all three genes is differentially activated during embryonic epidermal stratification. Manic-fng and Lunatic-fng are expressed in the basal layer, whereas Lunatic-fng is expressed in the granular layer and Radical-fng is restricted to the most differentiated nucleated layer. This expression decreases by a few days postnatally and can be reactivated by retinoic acid treatment, which triggers a new distribution of Fringe transcripts and a thickening of the granular layer. Therefore, Manic, Lunatic, and Radical Fringe by modulating the Notch pathway may play a key role in defining the different steps of keratinocyte differentiation.

CHOXC-8 and CHOXD-13 expression in embryonic chick skin and cutaneous appendage specification.

We studied the expression of two distantly clustered Hox genes which could, respectively, be involved in specification of dorsal feather- and foot scale-forming skin in the chick embryo: cHoxc-8, a median paralog, and cHoxd-13, located at the 5' extremity of the HoxD cluster. The cHoxc-8 transcripts are present at embryonic day 3.5 (E3.5) in the somitic cells, which give rise to the dorsal dermis by E5, and at E6.5-8.5 in the dorsal dermal and epidermal cells during the first stages of feather morphogenesis. The cHoxd-13 transcripts are present at E4.5-9.5 in the autopodial mesenchyme and at E10.5-12.5 in the plantar dermis during the initiation of reticulate scale morphogenesis. Both the cHoxc-8 and cHoxd-13 transcripts are no longer detectable after the anlagen stage of cutaneous appendage morphogenesis. Furthermore, heterotopic dermal-epidermal recombinations of dorsal, plantar, and apteric tissues revealed that the epidermal ability or inability to form feathers is already established by the time of skin formation. Retinoic acid (RA) treatment at E11 induces after 12 hr an inhibition of cHoxd-13 expression in the plantar dermis, followed by the formation of feather filaments on the reticulate scales. When E7.5 dorsal explants are treated with RA for 6 days, they form scale-like structures where the Hox transcripts are no more detectable. Protein analysis revealed that the plantar filaments, made up of feather beta-keratins, corresponded to a homeotic transformation, whereas the scale-like structures, composed also of feather beta-keratins, were teratoid. These results strengthen the hypothesis that different homeobox genes play a significant role in specifying the regional identity of the different epidermal territories.

Virulence of Aspergillus fumigatus strains investigated by random amplified polymorphic DNA analysis.

A possible relationship between the ability of Aspergillus fumigatus strains to invade tissues and genetic polymorphism was studied by random amplified polymorphic DNA (RAPD) analysis. One hundred randomly designed oligonucleotide decamers were examined with DNA of three reference strains, eight environmental isolates and 21 isolates from two distinct clinical situations: non-invasive aspergillosis (predominantly aspergilloma) and invasive aspergillosis. One primer (OPQ 6) was found to generate a reproducible amplification product that enabled distinction between the two groups according to the presence or absence of a 0.95-kb fragment that correlated with the nature of the infection (non-invasive or invasive) and immune status of the patient. The results indicated that the pathogenicity of A. fumigatus was related not only to the host's immune status but also to the virulence of the strain of A. fumigatus.

Identification of differentially transcribed RNA and DNA helicase-related genes of Plasmodium falciparum.

Chloroquine antimalarial action was assessed by the analysis of changes in gene expression. With this aim, Plasmodium falciparum cultures were submitted to chloroquine and to other stresses to determine which transcripts were specifically induced. P. falciparum in vitro control culture was compared to cultures where chloroquine was added and to cultures where serum was omitted, or where higher partial oxygen pressure was used, and, finally, at a temperature of 40 degrees C instead of 37 degrees C. Poly (A)+RNAs were reverse-transcribed and detected by the differential display technique. Two specific cDNAs were obtained and cloned, and a part of the genes was sequenced. The deduced protein, referred to as Pfhel-1, was related to a RNA helicase and was thought to be involved in protein translation control. The second deduced protein, called Pfhel-2, possessed consensus sequences of ATP-dependent helicase domains. Pfhel-2 may be involved either in mitotic control or in DNA repair. The possible roles of both helicase-related genes in chloroquine therapeutic activity are discussed.

Evidence for expression of a Ras-like and a stage specific GTP binding homologous protein by Plasmodium falciparum.

Plasmodium falciparum, the parasite responsible for the most severe form of malaria, undergoes an asexual multiplication in man and a sexual one in mosquito. The asexual cycle can be reproduced in vitro. The present work reports the isolation of a small guanosine triphosphate-binding protein in Plasmodium falciparum extracts. This protein, a 21,000 M(r) Ras-like molecule, was revealed by western blotting in each stage of the intraerythrocytic asexual life cycle. Conversely, a 46,000 M(r) G alpha subunit of a heterotrimeric GTP-binding protein was found to be expressed during a short period from mature schizonts to free merozoites. In order to provide additional evidence for the presence of these GTP-binding proteins in Plasmodium falciparum cultures and also to determine the kinetics, we tested two toxins that are involved in the cellular signalling transduction. We observed that pertussis toxin increases P. falciparum growth, whereas cholera toxin induces crisis forms, and subsequent parasite death within the following 24 h.

Development of natural immunity in Plasmodium falciparum malaria: study of antibody response by Western immunoblotting.

A longitudinal study was carried out in Burkina Faso to investigate the natural development of the immune response to Plasmodium falciparum malaria. Three bleedings were carried out before, during, and after the seasonal peak of transmission. Detailed antigen mapping and antibody prevalence of the 248 collected serum samples were established by immunoblotting on the basis of several epidemiological and biological parameters. An improved Western immunoblotting system was used to analyze up to 67 serum samples on each nitrocellulose sheet. This system allowed us to perform the entire study with strictly comparable conditions. Two different blood-stage antigens (exoantigens and somatic antigens) were used to analyze the distribution of different classes and subclasses of immunoglobulins according to the age of the individuals, the presence or absence of a malarial attack, the transmission period, the origin of parasite isolates, and the response to intraerythrocytic stages. Although this analysis emphasizes strong individual variations, reactions with two major antigens of 115 and 103 kDa were especially noted. These antigens induced high antibody levels and prevalences but were probably not involved in protection. The prevalence of immunoglobulin G (IgG) antibodies differed by isotype. Most of antigens stimulating IgG production were also responsible for the IgM antibody response. The role played by these antibodies in the development of natural immunity against malaria is discussed.

[Detection of homologous oncogene sequences in the genome of Plasmodium falciparum]. / Détection de séquences homologues d'oncogènes dans le génome de Plasmodium falciparum.

Homologous sequences of the acute RNA tumor virus oncogenes have been found to be highly conserved within vertebrates, insects and yeasts. In the present work, seven different oncogene DNA sequences have been used as probes to search for homologous sequences in the DNA of the protozoan Plasmodium falciparum. Both the v-fms v-Ha ras probes hybridized P. falciparum DNA. The oncogene study will allow an understanding of the biology of the parasite and particularly the host-parasite relationships which allow P. falciparum to develop, keeping the established harmony between the parasite and his host.

Purification of Plasmodium falciparum exoantigens by spun-column chromatography and by inverse affinity chromatography.

Exoantigens released into the medium during the in vitro culture of Plasmodium falciparum were purified to obtain a better characterization. This purification was done in two steps. First the crude culture medium was passed through a cationic exchange gel on a spun-column. This preliminary step is efficient and rapid. The serum contaminants were removed from the effluent by an inverse affinity chromatography with, as adsorbant, anti-human serum from the horse. The quality of the antigens obtained was analysed by metabolic radioactive labelling with tritiated leucine and by exclusion high pressure liquid chromatography, and the antigenicity measured by ELISA.

Trans-elution: a method for the purification of components of complex biological extracts.

Trans-elution is a simple and rapid technique allowing the purification of macromolecules trapped in polyacrylamide gels following electrophoresis. This method of purification consists of the sideways transfer of the macromolecules under the influence of an electric field from the gel slab toward an inert support. In contrast to the role of nitrocellulose used in the "Western blotting" technique, in this case the support does not bind the macromolecules. It consists of a network capable of retaining the buffer by capillary. The electroeluted proteins remain in solution in the buffer and it is thus easy to recover them by spin-drying the support. The support material is either glue-free paper or glass fiber paper. Small concentrated samples are obtained at high yield. The procedure does not require gel slicing, thus avoiding both manipulation of the gel and errors in the localization of the fractions to be purified. In a single step all the proteins fractionated on the gel may be eluted. The trans-elution technique has been applied to the purification of Plasmodium falciparum and Toxoplasma gondii antigens, the causative agents of malaria and toxoplasmosis, respectively.

A septate polycarbonate cell culture unit used for Plasmodium falciparum and hybridomas.

A new material, makrolon, is used for the construction of a large-scale cell culture vessel. It is strong, light, transparent, thermostable, septate and inexpensive. Several independent vessels of 500 ml each can be stacked. It has been used for Plasmodium falciparum and hybridoma cultures, where frequent renewal of the medium and a large gas/liquid interface are required.

[New prospects for the epidemiologic and immunologic study of malaria by the Western blotting technic]. / Perspectives ouvertes à l'étude épidémiologique et immunologique du paludisme par la technique d'immuno-transfert.

A new modification in western blotting technique now permits the analysis of micro samples of blood collected from inhabitants of malaria endemic areas using several different antigens of Plasmodium falciparum. Compared to Immuno Fluorescent Assay and to Immuno Enzymology, ELISA (using somatic antigens and exoantigens of P. falciparum) the western blotting method gives a more detailed analysis. It seems to open new prospects for seroepidemiological studies of human malaria and for the selection of antigenic fractions that may allow the preparation of a vaccine.

Purification and immunochemical study of Plasmodium falciparum exoantigens.

Plasmodium falciparum, in in vitro culture, elaborated many antigens including soluble exoantigens that are released into the culture medium. Anionic and cationic methods of isolating these antigens offer a great potential for large scale purification from medium that is rich in proteins but contains relatively low concentrations of P. falciparum specific antigens. These exoantigens have cationic and anionic dependent elution profiles (pI between 3.7 and 4.8). Five apparent molecular weight entities (58, 80, 145, 200, and 290 kdaltons) have been determined by GEDELISA. Susceptibility to lipase and to a proteolytic enzyme confirmed the proteinaceous nature of the antigens. They were isolated from 4 strains of different geographic origin, indicating their ubiquitous nature. The analogy of these exoantigens to circulating antigens in patients with acute malaria and their potential usefulness in immunodiagnosis and immunoprophylaxis are discussed.

Incipient resistance of Plasmodium falciparum to chloroquine among a semi-immune population of the United Republic of Tanzania. 2. The impact of chloroquine used as a chemosuppressant on the immune status of the population.

Decreased sensitivity and incipient resistance of Plasmodium falciparum strains to chloroquine have been reported from Mto-wa-Mbu, in the north-east of the United Republic of Tanzania. In this locality the population had been exposed to chloroquine pressure for about two decades, in the form of medicated salt and through easy availability of the drug itself. In an attempt to find out whether such chemosuppression had influenced the immune response of the population, two seroepidemiological surveys were carried out in March 1981 and March 1982; the second survey was performed to confirm the results obtained in the first one. The humoral immunological response was measured by the immunofluorescent antibody technique. In the absence of information on the immunological profile that existed in the area prior to the introduction of chloroquine in 1960, the results of the present surveys were compared with those obtained in another locality in the north-east of the United Republic of Tanzania in 1967, and in the West Kiang district of Gambia in 1965. The two areas used for comparison exhibited a malaria endemicity similar to that prevailing in Mto-wa-Mbu prior to the introduction of the medicated salt. The results from Mto-wa-Mbu showed a significantly lower proportion of subjects with positive titres and a lower geometric mean titre in all age groups.A reduction in the humoral immunological response might be explained by the drug pressure that has been exerted in the area for many years. The depressed immune response found at Mto-wa-Mbu, however, was so marked that other factors may have contributed to its establishment.In view of the importance of these findings, it is recommended that further, longitudinal serological studies be conducted in the field to assess the effects of chemosuppression on the immune response of the protected populations.