The involvement of the trans-generational effect in the high incidence of the hydatidiform mole in Africa.

INTRODUCTION: While the incidence of various chromosomal anomalies observed, including triploid partial moles is independent of the socio-economic level, higher incidences of complete hydatidiform mole "CHM" is generally associated with under developed areas. Moreover, studies have shown that some nutritional deficiencies are related to the abnormal development of oocytes and placenta. In Senegal and Morocco, the annual seasonal cycle contains one period with food shortages and the incidence of complete moles is significant. Accordingly, accurate statistical analyses have been performed in these two countries. METHODS: Each month during a one year period, we investigated the occurrence of normal conceptions, molar conceptions and the conception of the future patients in Senegal and Morocco. The comparisons of the conception dates for these three types of conception were analyzed using the Chi-squared test. RESULTS: 94% of the patients were conceived just prior to the period in the year with food shortages. Consequently, the development of the female embryos occurred under nutritional constraints, which negatively affect the recruitment of the vital factors required for the normal synthesis of DNA, proteins and placental differentiation. DISCUSSIONS: A nutritional deficiency in the mother at conception of their daughter (future patient) is implicated in the higher incidence of CHM in their daughters' filiation. These nutritional deficiencies during the first weeks of pregnancy will have repercussions on the normal development of the oocytes. Accordingly, these developmental impairments take place during the embryonic life of the future mothers of complete moles and not during the conception of the moles themselves.

Cytogenetics, conserved synteny and evolution of chicken fucosyltransferase genes compared to human.

Fucosyltransferases appeared early in evolution, since they are present from bacteria to primates and the genes are well conserved. The aim of this work was to study these genes in the bird group, which is particularly attractive for the comprehension of the evolution of the vertebrate genome. Twelve fucosyltransferase genes have been identified in man. The orthologues of theses genes were looked for in the chicken genome and cytogenetically localized by FISH. Three families of fucosyltransferases: alpha6-fucosyltransferases, alpha3/4-fucosyltransferases, and protein-O-fucosyltransferases, were identified in the chicken with their associated genes. The alpha2-fucosyltransferase family, although present in some invertebrates and amphibians was not found in birds. This absence, also observed in Drosophila, may correspond to a loss of these genes by negative selection. Of the eight chicken genes assigned, six fell on chromosome segments where conservation of synteny between human and chicken was already described. For the two remaining loci, FUT9 and FUT3/5/6, the location may correspond to a new small syntenic area or to an insertion. FUT4 and FUT3/5/6 were found on the same chicken chromosome. These results suggest a duplication of an ancestral gene, initially present on the same chromosome before separation during evolution. By extension, the results are in favour of a common ancestor for the alpha3-fucosyltransferase and the alpha4-fucosyltransferase activities. These observations suggest a general mechanism for the evolution of fucosyltransferase genes in vertebrates by duplication followed by divergent evolution.

Use of embryonic human trachea grown in nude mice to patch-repair congenital tracheal stenosis.

BACKGROUND: Long congenital tracheal stenosis is a life-threatening condition, and the available surgical treatments do not give satisfactory long-term results. METHODS: Human embryonic tracheas were implanted in the abdominal cavities of nude mice until their differentiation was completed. These differentiated tracheas were used to patch-repair surgically induced tracheal stenosis in piglets. The human, mouse, or pig origin, of all the cells in the two successive xenotransplants in the nude mouse and the pig, was determined on tissue sections by in situ hybridization with species-specific DNA probes. RESULTS: The transplanted pigs thrived and reached normal adulthood, irrespective of the administration of immunosuppressive treatment. The human tracheal tissue developed in nude mice conserved human structures, with the exception of feeding capillaries, which were of mouse origin. The tracheal patch in the adult healthy pigs comprised only pig cells organized into a fibrous scar, which was covered by normal pig epithelium. CONCLUSIONS: Results suggest that human embryonic trachea grown in nude mice can be successfully used as patch tracheoplasty for long congenital tracheal stenosis without conventional immunosuppression.

Demonstration of human kidney differentiation antigens with monoclonal antibodies.

Six human differentiation antigens (EE24.6, EG9.11, EG14.1, EI16.1, EK8.1, EK17.1) have been defined using monoclonal antibodies obtained from mice immunized with embryonic kidney cells. Their histologic distribution was determined on frozen sections of embryonic, fetal, and adult human kidneys by immunofluorescence assay. EE24.6, an ureteral bud marker, was detected only on the germ layer of mature kidney urothelium. EG9.11 and EG14.1 were detected on the S-shaped bodies and also on the adult proximal convoluted tubule for the former and the glomerular basement membrane for the latter. EI16.1, a marker of condensed mesenchyme, was detected only on epithelial cells of adult proximal convoluted tubule. EK8.1 was found in the mesangium, connective tissue, and with particularly dense labeling in the basement membranes. This labeling pattern was present throughout renal organogenesis. EK17.1 recognized both cell and plasma human fibronectins. Staining for all antibodies was nearly identical in mesonephros and metanephros. These results demonstate that some antigens follow their embryonic destiny. They indicate an antigenic similarity between the mesonephros and the metanephros and, therefore, a very early appearance of these antigens. During differentiation, these antigens concentrate on more defined structures, and staining became increased with an increased degree of differentiation.

Five specificity patterns of (1----3)-alpha-L-fucosyltransferase activity defined by use of synthetic oligosaccharide acceptors. Differential expression of the enzymes during human embryonic development and in adult tissues.

The use of synthetic trisaccharides as acceptors led to the definition of five main (1----3)-alpha-L-fucosyltransferase activity patterns in human adult tissues: (I). Myeliod cells, granulocytes, monocytes, and lymphoblasts, transfer an alpha-L-fucopyranosyl group to O-3 of a 2-acetamido-2-deoxy-D-glucosyl residue of H blood-group Type 2 oligosaccharide [alpha-L-Fucp-(1----2)-beta-D-Galp-(1----4)-beta-D-GlcpNAc----R] with Mn2+ as activator. (II) Brain has the same acceptor specificity pattern as myeloid cells, but can also use Co2+ as activator. (III) Plasma and liver transfer an alpha-L-furopyranosyl group to H blood-group Type 2 and to sialyl-N-acetyllactosamine [alpha-NeuAc-(2----3)-beta-D-Galp-(1----4)-beta-D-GlcpNAc----R]. (IV) Intestine, gall bladder, kidney, and milk have the same activity as (III), but also transfer an alpha-L-fucopyranosyl group to O-4 of a 2-acetamido-2-deoxy-D-glucose residue of H blood-group Type 1 [alpha-L-Fucp-(1----2)-beta-D-Galp-(1----3)-beta-D-GlcpNAc----R] and sialyl Type 1 [alpha-NeuAc-(1----3)-beta-D-Galp-(1----3)-beta-D-GlcpNAc----R]. (V) Stomach mucosa is not able to use sialyl-N-acetyllactosamine, but can transfer an alpha-L-fucopyranosyl group to the other Type 1 and Type 2 acceptors. Unlike in adult tissue, a single myeloid-like pattern of (1----3)-alpha-L-fucosyltransferase activity was found at early stages of development in all tissues tested. This embryonic enzyme is later progressively replaced by enzymes or mixtures of enzymes having the corresponding adult patterns of enzyme expression. All lymphoblastoid cell lines and half of the tumor epithelial cell lines tested expressed the myeloid-like pattern of enzyme found in normal embryonic tissues. The remaining tumor epithelial cell lines expressed different forms of (1----3/4)-alpha-L-fucosyltransferase acceptor specificity patterns.

Molecular genetics of alpha-L-fucosyltransferase genes (H, Se, Le, FUT4, FUT5 and FUT6).

Six human alpha-L-fucosyltransferase genes have been registered in the GDB as FUT1 to FUT6 according to the chronology of their description. FUT1 and FUT2 encode the alpha(1,2)fucosyltransferases H and Se respectively. The FUT2 gene has not been cloned, but it is expected to be closely linked to FUT1 on the long arm of chromosome 19. FUT3, FUT4, FUT5 and FUT6 encode different alpha(1,3)fucosyltransferases which share between 60 and 90% homology with each other, but none with FUT1. Missense and nonsense point mutations have been found to inactivate the cognate enzymes of FUT1, FUT3 and FUT6. FUT3 and FUT6 are closely linked on the short arm of chromosome 19 and encode the Lewis and plasma enzymes respectively. The FUT5 gene has been cloned and sequenced, but its tissue expression has not been defined as yet. FUT4 has been mapped to 11q21 and encodes a monomorphic myeloid enzyme. All but FUT4 are genetically polymorphic. The deficient alleles of FUT1 and FUT6 have a very low incidence and they have been found mainly around the Indian Ocean. A myeloid enzyme is present in 5 to 10 week old human embryos and is later progressively replaced by different patterns of adult fucosyltransferase enzymes in all tissues, except in leukocytes and brain which continue to express a FUT4 like enzyme in the adult.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytogenetic repartition of chicken CR1 sequences evidenced by PRINS in Galliformes and some other birds.

Chicken repeat 1 (CR1) belongs to the non-long repeat class of retrotransposons. Nearly 100000 repeats interspersed in the chicken genome are subdivided into at least six distinct subfamilies, each 300 bp long and all sharing substantial sequence similarity. CR1-like elements were found in genomes from invertebrates to mammals, suggesting their importance for genome structure and/or function. Moreover, numerous data support the hypothesis of their implication in regulation of gene expression. So, the chromosomal distribution of these CR1 sequences in vertebrates is of great interest to improve our knowledge about the genome structure, function and evolution. A comparison of the cytogenetic distribution of CR1 sequences was performed by PRINS using consensus chicken primers on the chromosomes of chicken and species of several bird orders: Galliformes, Anseriformes, Passeriformes and Falconiformes. The study revealed that CR1 repeats are spread over nearly all chicken chromosomes with a higher density on the macrochromosomes and in particular with hot spots on subtelomeric regions of chromosome 1, 2, 3q, 4q, 5q. Their distribution on the macrochromosomes forms a kind of banding pattern, which was not systematically matched with R- or G-banding. This banding pattern appears to be conserved on the chromosomes of the Galliformes studied, irrespective of their karyotypes, rearranged or not. CR1 primers also show similar signals on the chromosomes of birds phylogenetically more distant (Anseriformes, Passeriformes and Falconiformes). This fact confirms the importance of these sequences at the large scale of bird evolution and in the chromosomal structure. The location of CR1 sequences, and in particular of the hot spots, mainly within the richest CG areas are in conformity with the data on an epigenetic role of these highly conserved sequences.

Molecular genetics of H.

BACKGROUND AND OBJECTIVES: Formal genetics of ABO, H-h and Se-se systems illustrate that these three systems are genetically independent MATERIALS AND METHODS: Population analysis of phenotypes and family segregation of the ABH related genetic markers RESULTS: Inactivating mutations of FUT1 and FUT2 are compatible with a structural gene model assuming that FUT1 and FUT2 genes encode for two distinct enzymes, one encoding for the H antigen expressed in red cells (FUT1) and the other encoding for the H gene expressed in saliva (FUT2) CONCLUSION: Most inactivating mutations of FUT1 and FUT2 genes are located in the coding region of the genes and are nonprevalent sporadic mutations of relative recent appearance.

[Antigenic study of the differentiation of the human kidney using monoclonal antibodies]. / Etude antigénique de la différenciation du rein humain par des anticorps monoclonaux.

The production of monoclonal antibodies against human embryonic renal cells allowed to display on the adult human kidney some antigens typical of certain structures or tissues: the proximal convoluted tubule for EG 9-11 and EG 19-6 monoclonal antibodies, the glomerular basement membrane for EG 14-1, the urothelium for EE 24-6, the connective tissue for EK 8-1 and EK 17-1 and probably the capsular and tubular basement membranes for EK 8-1. Simultaneously, we could follow the spatial and temporal repartition of the antigens during the renal development. One of them (EI 16-1) seemed to disappear in the adult and might correspond to a foetal type-antigen.

[Determination of cell surface antigens on cryostat sections with monoclonal antibodies]. / Mise en évidence des antigènes de surface cellulaire sur des coupes au cryostat grâce à des anticorps monoclonaux.

Most monoclonal antibodies recognize antigens which do not survive conventional tissue processing: the use of frozen tissue sections and the immunofluorescence method overcome this obstacle but introduce other problems. Three improvements are reported here: the use of serum-free (substitute) "Ultroser Hy" as a culture medium for hybridomas, in order to diminish background staining and the diffusion artifact; the use of freon for freezing tissue sections, so as to slightly increase cellular morphology and staining; the use of a new immunofluorescent slide-mounting medium to enhance histologic preservation and immunohistologic contrast and to diminish fading of immunofluorescence.

[Tissue expression of a gene potentially implicated in some diseases with retinal and renal involvement]. / Expression tissulaire d'un gène potentiellement impliqué dans certaines maladies associant une atteinte rétinienne et rénale.

We have produced a monoclonal antibody (HA34) that specifically reveals the pigmented epithelium in the eye and the proximal convoluted tubules of the kidney, whatever the developmental stage. The results obtained with the kidney of other mammals suggest that the antigen is human specific. Its molecular weight is approximately 200 kDa. The epitope recognized by HA34 is always present on cell lines grown in vitro. This allowed us to use somatic cell interspecific hybrids to localize the gene implicated in the cytogenetic band 11q13, between microsatellites D11S1777 (AFMa046wa9) and D11S913 (AFM164zf12) in a 9 cM space. This region is involved in forms of retinitis pigmentosa, some of which can also include kidney abnormalities. We propose that this gene is possibly implicated in some of these diseases.

Expression of fucosyltransferases in skin, conjunctiva, and cornea during human development.

During human development, type-1-precursor, sialyl-Le a, and Le x antigens were present in the periderm of skin and eye at week 6. The Le x antigen disappeared from cornea at 10 weeks and then from skin at 20 weeks. H-type-1, Le a, Le b, sialyl-Le a, H-type-2, sialyl-Le x, and Le y were found in cornea, conjunctiva, and periderm between 10 and 20 weeks. They disappear from the skin (at week 20) and progressively reappear in skin derivatives, especially in the epithelium of sweat glands. The secretory part of the sweat gland is type-1-precursor and H-type-1 positive while its excretory part is Le a, Le b, sialyl-Le a, and Le y positive. On the eye surface the disappearance of Le x at 10 weeks and of the H-type-1, sialyl-Le x, and Le y at week 35 starts in the central cornea in front of the lens. The corneal epithelium and the conjunctiva have similar antigens to those of excretory and secretory parts of the sweat gland, respectively. Invaginations and folding of the epidermis might preserve the embryonic staining. We propose that fucosylation patterns are associated with the embryonic origin and differentiation stage of tissue. The early and transient presence of Le x is associated with FUT4 or FUT9 activities, while the late appearance of Lewis antigens is related to other alpha3-fucosyltransferases.

Developmental modifications of sheep kidney antigens.

We have used monoclonal antibodies to study the changes in the expression of four kidney antigens during organogenesis in the sheep. Two of these antibodies, EE24.6 and EJ30.1, label intensely only the adult kidney, whereas the other two, EK17.1 and EJ15.1, bind to the extracellular matrix of the embryonic kidney. For EJ15.1, the staining of the extracellular matrix decreases temporarily during the second half of intrauterine life, a period during which a light staining appears in the mesangium. For the other, EK17.1, the extracellular matrix staining in the stroma gradually decreases as the embryo grows, while staining of the mesangium and the arterial intima becomes evident. With EK17.1, fibronectin is identified in the extracellular matrix of the embryonic kidney and intracellularly in the mesangial cells after these cells have colonized the glomerulus. The mesonephros staining seems to be the same as that of the metanephros. In the adult, extraglomerular vascular endothelial cells bind EK17.1, whereas intraglomerular endothelial cells do not express fibronectin, which suggests a functional difference between endothelial cells in these two localizations.

[Phylogenic distribution of human renal antigens defined by monoclonal antibodies]. / Etude de la répartition phylogénique d'antigènes rénaux humains définis par des anticorps monoclonaux.

The preparation fo five monoclonal antibodies specific of important human renal histologic structures both functionally and organogenetically has permitted to identify the repartition of the corresponding antigens in the vertebrate phylum. For three of them, appeared a clear cut histologic identity in intensity and localization between the mammals studied and man. For the two others a phylogenic and histologic dispersion was observed. It may be supposed, in the latter case, that the evolution and the biotope have acted in different manners on renal function and organogenesis according to the vertebrate classes or species investigated.

Use of monoclonal antibodies to follow the phylogenic distribution of human renal antigens.

Nine human differentiation antigens have been defined by monoclonal antibodies (M. Abs) developed from mice immunized with embryonic kidney cells (mesonephros or metanephros of 7 week-developmental ages). Their spatial and temporal distributions during human kidney organization were previously studied [3]. In this paper we have attempted to follow by immunofluorescence their phylogenic location, from fish to mammals. Six of them recognized the same structures as in humans: proximal convoluted tubules (PCT) (EG9.11, EG19.6, E116.1), glomerular basement membrane (GBM) (EG14.1) and extracellular matrix (EK8.1, EK17.1). However, staining was limited to certain mammals. EK17.1 has been characterized as an anti-fibronectin. These antibodies revealed the same histological structures in the human mesonephros and metanephros. The three other antibodies revealed epitopes appearing earlier in evolution and whose histological distribution varied according to species. These antibodies stained different structures in the mesonephros and metanephros. Thus, the staining particularities observed during human renal ontogenesis were found again in the phylogenetical study.

Genetic regulation of the expression of ABH and Lewis antigens in tissues.

Sequential appearance of ABH antigens in different animal species shows a progression from tissues of endodermal to ectodermal and finally mesodermal origin, human erythrocytes being the last cells to acquire these antigens. In view of this, ABH antigens should be called tissue or histo-blood group antigens rather than blood group antigens. In addition to the glycosyltransferases encoded by the ABO genes, several alpha-2, alpha-3 and alpha-4-fucosyltransferases are needed to account for the known ABH histo-blood group antigens. The genetic polymorphism of the genes encoding each of these enzymes defines inter-individual differences. In addition, in the same individual various tissues express these antigens in a different way. For each adult epithelial tissue, antigenic expression is related to cell maturation from germinal layer to surface epithelium. Differential expression is also found at various embryonal stages of the same cells. Examples of these phenomena are presented in an effort to gain further insight into the genetic regulation of the expression of these complex oligosaccharide molecules.

Primed in situ (PRINS) labelling with Alu and satellite primers for rapid characterization of human chromosomes in hybrid cell lines.

The primed in situ (PRINS) labelling method was developed as an alternative to classical cytogenetics and fluorescence in situ hybridization (FISH) for the characterization of interspecific somatic hybrids. Full karyotypes were performed by PRINS using Alu-specific primers to generate the painting of all human material associated with R-like banding. The representativity of individual human chromosomes was established using primers specific for discriminent alpha-satellite DNA sequences providing specific signals on the centromeres of the targeted chromosomes and corresponding spots in interphase nuclei. Using this methodology, a somatic hybrid clone was shown to be monochromosomal for the der(11) from a t(11;22) patient.

[Rapid identification of chromosomes by in situ hybridization of labelled oligonucleotides and comparison with the PRINS method]. / Identification rapide des chromosomes par hybridation in situ d'oligonucléotides marqués et comparaison avec la méthode PRINS.

We propose a simple, fast and inexpensive method of identification of human centromeres on metaphasic chromosomes and interphasic nuclei. This is based on in situ hybridization of labelled oligonucleotides. The efficiency of the methodology was demonstrated on cytogenetic preparations from human heteroploid and human x hamster hybrid cell lines and also on frozen tissue sections using an oligonucleotide specific for the alpha-satellite DNA of chromosome 1. Three versions of this oligonucleotide respectively labelled with 1, 4 and 10 fluorescein molecules were synthesized. The signal intensity provided by the oligonucleotide coupled with 4 fluoresceins allowed unambiguously the detection of the chromosome and the establishment of its ploidy using a classical cytogenetic microscope without the need for an amplification procedure. The use of different fluorochromes and possibly combination with an unlabelled elongation in 3' of the oligonucleotides which stabilize its hybridization, lead to a simple multicolour method. Preliminary quantification of the signals obtained by in situ hybridization of labelled oligonucleotides and comparison with those obtained by primed in situ labelling (PRINS) using the same nucleotides as primers, suggest that the elongation generated by PRINS may be very short compared with a PCR in solution. This limited efficiency of the in situ elongation may reflect the present difficulties of PRINS and DISC PCR (direct in situ single copy polymerase chain reaction) with primers specific for non-repetitive sequencies.

Cytogenetic characterization of interspecific somatic hybrids by PRINS.

The primed in situ (PRINS) labeling technique was developed as an alternative method to classical cytogenetics and in situ hybridization (FISH) for the characterization of interspecific somatic hybrids. Full karyotypes were performed using Alu specific primers generating the painting of all human material associated with R like banding. The representativity of individual human chromosomes was established using primers specific for discriminent alpha-satellite DNA sequences providing specific signals on the centromeres of the targeted chromosomes and corresponding spots in interphase nuclei. Due to the use of synthetic oligonucleotide primers and of directly labeled haptens. PRINS method avoid repetitive probes preparation, eliminates secondary amplification of signals and the whole process can be performed within a timespan of 1 hour. Providing qualitative and quantitative answers, the simple PRINS method appears very well adapted to the specific problematic of somatic hybrids as for their characterization than for their periodic controls imposed by their instability. The method has been tested on 4 human-rodent hybrid cell lines. In particular, the somatic hybrid clone ALE 4 was shown to be monochromosomal for the der(11) from the reciprocal translocation t(11:22).