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Biomedical subjects

A Forabosco

Publications and source records attributed to A Forabosco.

At least 37 records · Page 2Linked to original sources
PubMed1999

Fetal erythroblast isolation up to purity from cord blood and their culture in vitro.

BACKGROUND: Erythroblasts have been the most encouraging candidate cell type for noninvasive prenatal genetic investigation. We previously showed that human erythroblasts can be recovered from bone marrow and blood bank buffy coats by a physical cell separation. In the present study, we modified our previous methodology, taking into account the peculiar behavior of erythroblasts in response to modifications of pH and osmolality of the separation medium. METHODS: Twenty to forty milliters of cord blood were initially centrifuged on Ficoll/diatrizoate (1.085 g/ml). The interphase cells were further separated on a continuous density gradient (1.040-1.085 g/ml). Two different gradients were initially compared: the first was iso-osmolar and neutral, whereas the second also contained an ionic strength gradient and a pH gradient (triple gradient). A subsequent monocyte depletion was performed by using magnetic microbeads coated with anti-CD14 monoclonal antibody (mAb), and erythroblasts were purified by sedimentation velocity. Purified cells were investigated by analyses with fluorescence-activated cell sorting (FACS) and fluorescence in situ hybridization (FISH) and immunocytochemistry with mAb against fetal hemoglobin and were cultured in vitro. RESULTS: When nucleated cells were spun on an iso-osmolar and neutral continuous density gradient, two separated bands of nucleated red blood cells (NRBCs) were obtained: a light fraction banding at 1.062 g/ml and an heavy fraction banding at 1.078 g/ml. Conversely, when cells were spun in the triple gradient, NRBCs were shifted to the low-density region. Monocyte depletion by immunomagnetic microbeads and velocity sedimentation provided a pure erythroblast population. FACS and FISH analyses and immunocytochemistry substantiated the purity of the isolated cell fraction, which was successfully cultured in vitro. CONCLUSIONS: We have shown that fetal erythroblasts can be purified up to homogeneity from cord blood, but further refinements of the isolation procedure are necessary before the same results can be obtained from maternal peripheral blood.

Blood Sedimentation↗
PubMed1999

First-trimester fetal sex prediction by deoxyribonucleic acid analysis of maternal peripheral blood.

OBJECTIVES: We investigated whether the number of weeks of gestation influences the accuracy of first-trimester fetal sex prediction by analysis of deoxyribonucleic acid extracted from whole maternal blood. A comparison was also made to determine whether a difference exists between this approach and the deoxyribonucleic acid analysis of transcervical cells performed on the same group of subjects. STUDY DESIGN: Deoxyribonucleic acid was isolated from 50 maternal blood samples taken between gestational weeks 7 and 11. The sex of the fetus was assessed by nested polymerase chain reaction specific for the amelogenin gene. A receiver-operating characteristic curve analysis was used to correlate the accuracy of fetal gender prediction with the gestational age and also to compare the goodness of the 2 methods under investigation. RESULTS: Analysis of the receiver-operating characteristic curve provided a cutoff value of 9 weeks 4 days of gestation for both tests, indicating that a higher degree of accuracy in the sex assignment was obtained in those samples taken before or at this time. However, this difference was statistically significant only for analysis of deoxyribonucleic acid from maternal blood. The comparison between tests of deoxyribonucleic acid from maternal blood and from transcervical cells showed that the first approach is better, although a statistically significant difference was not found. CONCLUSION: Analysis of maternal blood deoxyribonucleic acid is a better approach than analysis of trans-cervical cell deoxyribonucleic acid in fetal sex prediction. The highest degree of accuracy is obtained when blood is drawn before 10 weeks of gestation. This can be important when sampling of chorionic villi should be avoided because of the risk of an X-linked disease when the fetal sex is female.

Amelogenin↗
PubMed1999

Biometrical threshold of biparietal diameter for certain fetal sex assignment by ultrasound.

OBJECTIVES: The aim of this study was to establish the biometric threshold of biparietal diameter (BPD), assumed to be an independent variable of gestational age, at which 100% accuracy in the assessment of fetal sex by ultrasonography is achievable. METHODS: Transvaginal and/or transabdominal sonography was used for detecting the 'sagittal sign' as a marker of fetal sex in 385 fetuses with BPD between 18 and 29 mm. The results of ultrasound examination were compared with sex at birth or with karyotype obtained from amniotic fluid cells or chorionic villus sampling. RESULTS: Fetal sex assignment was feasible in 337 of 385 cases (87.5%). Of the 312 fetuses with known fetal sex outcome, 164 were males and 148 were females. An accuracy rate of 100% was achieved when a BPD of > or = 23 mm was obtained. CONCLUSION: This study provides important information about the earliest stage of fetal development, expressed in terms of BPD, at which a diagnosis of fetal sex can be made with 100% accuracy.

Biometry↗
PubMed1999

A novel pseudoautosomal human gene encodes a putative protein similar to Ac-like transposases.

We report the cloning of a novel gene, called Tramp, in the Xp/Yp PAR region that has a functional homologue on the Y chromosome and escapes X-inactivation. This gene encodes, within a single exon, a putative protein that has amino acid similarity with transposases of the Ac family. Flanking this gene we have identified putative terminal inverted repeats (TIRs) and a duplicate target site, suggesting that it may be an ancient transposable element. The nucleotide differences in these sites and the TIR-binding inactivity of the putative Tramp protein suggest that this element is not an autonomous transposon. In the human genome, the Tramp protein may be involved in the transposition of other transposable elements, like medium reiterated frequency repeats, or it could be specialized in the acquisition of a new cellular function.

Amino Acid Sequence↗
PubMed1998

Timing of gene expression and oolemma localization of mouse alpha6 and beta1 integrin subunits during oogenesis.

The sperm antigen fertilin alpha/beta and the integrin complex alpha6beta1 present on the oolemma are two of the most promising candidates to mediate gamete interaction. During growth, the plasma membrane of both hamster and mouse zona-free oocytes acquires the capacity to fuse with acrosome-reacted sperm when oocytes reach the size of 25-30 microm in diameter, suggesting changes in the membrane molecular composition. The present study has two aims: to determine the timing of (1) gene expression of alpha6 and beta1 integrins and (2) localization of these integrin subunits on the plasma membrane in primordial germ cells and in oocytes during oogenesis. We found that both alpha6 and beta1 genes are expressed in female germ cells during all the stages of development analyzed, from 10.5 to 18.5 d.p. c., during oocyte growth, and in ovulated eggs. The alternatively spliced isoform alpha6B is expressed from 10.5 d.p.c., whereas alpha6A begins to be expressed at 12.5 d.p.c., suggesting a different role for the two variants. In situ immunodetection of alpha6 or beta1 shows a ring of fluorescence on the female germ cell plasma membrane for both integrins at 10.5 d.p.c., then the fluorescent signal becomes undetectable at 12.5 d.p.c. to reappear again, this time with a patchy distribution, at 18.5 d.p.c. This pattern of localization is maintained in oocytes isolated from newborn individuals and only when oocytes during growth reach the size of about 25-30 microm in diameter does the fluorescence become homogenous all around the whole oocyte surface. These data, although not conclusive, support the hypothesis of an involvement of alpha6 and beta1 integrins in sperm-egg fusion.

Animals↗
PubMed1998

Barber-Say Syndrome: report of a new case.

We present a girl with lax, redundant skin, ectropion, bulbous nose, macrostomia, and absence of mammary glands. To our knowledge, she represents the fourth described case of Barber-Say Syndrome (BSS). BSS and ablepharon macrostomia syndrome (AMS) share common and distinctive clinical manifestations that involve the same structure of the skin and adnexa. We hypothesize that they may derive from a defective regulation of the same gene.

Abnormalities, Multiple↗
PubMed1998

Gpc3 expression correlates with the phenotype of the Simpson-Golabi-Behmel syndrome.

Interest in glypican-3 (GPC3), a member of the glypican-related integral membrane heparan sulfate proteoglycans (GRIPS) family, has increased with the finding that it is mutated in the Simpson-Golabi-Behmel overgrowth syndrome (Pilia et al. [1996] Nat. Genet. 12:241-247). The working model suggested that the membrane-bound protein acts locally to limit tissue and organ growth and that it may function by interacting with insulin-like growth factor 2 (IGF2) to limit its local effective level. Here we have tested two predictions of the model. In situ hybridization with the mouse gene cDNA was used to study the expression pattern during embryonic and fetal development. In agreement with predictions, the gene is expressed in precisely the organs that overgrow in its absence; and the patterns of expression of Gpc3 and those reported for Igf2 are strictly correlated.

Abnormalities, Multiple↗
PubMed1998

A novel pseudoautosomal gene encoding a putative GTP-binding protein resides in the vicinity of the Xp/Yp telomere.

We report the cloning of a novel Xp/Yp pseudoautosomal gene called PGPL , and demonstrate that PGPL , like other pseudoautosomal genes, escapes X inactivation and has a functional homologue on the Y chromosome. This gene is expressed in all the tissues examined and is highly conserved across several species. The PGPL gene encodes a protein of 442 amino acids and shows the consensus sequences of a series of motifs of the GTP-binding protein domain. Using fluorescence in situ hybridization analysis on normal males and on patients with rearrangements in the pseudoautosomal region, the gene was localized within 500 kb of the telomere. Further refinement using a cosmid contig of the region places this novel gene within 80-110 kb of the telomere, making this the most telomeric gene on the short arms of the sex chromosomes.

Amino Acid Sequence↗
PubMed1998

Meiotic and developmental competence of mouse antral oocytes.

Mouse antral oocytes show two different patterns of chromatin organization, defining oocytes with or without chromatin surrounding the nucleolus (SN: surrounded nucleus; NSN: not surrounded nucleus). We have previously shown that upon injection of eCG, NSN antral oocytes shift towards the SN kind of chromatin organization. We hypothesized that these newly formed SN oocytes were those that would have been ovulated after an ovulatory stimulus. The main objective of this study was to investigate the meiotic and developmental competence of these two types of oocytes. SN and NSN antral oocytes were isolated after i.p. administration of eCG + hCG or eCG-only, in vitro-cultured until completion of metaphase II, and inseminated with capacitated spermatozoa; and their development to the 4-cell stage was examined. This study demonstrates 1) that SN and NSN oocytes isolated after injection of eCG + hCG are capable of embryonic development, but not beyond the 2-cell stage; and 2) that SN and NSN oocytes isolated after injection of eCG-only are capable of developing to the 2-cell stage, but a significantly higher number (11.9%) of SN oocytes than NSN oocytes (1.5%) reach the 4-cell stage. SN- and NSN-like oocytes have also been described in the antral compartment of human, rat, monkey, pig, and bovine ovaries. The findings reported in this paper may contribute to improved procedures for in vitro fertilization of humans and farm animals.

Animals↗
PubMed1998

Mutation in the nerve-specific 5'non-coding region of Cx32 gene and absence of specific mRNA in a CMTX1 Italian family. Mutations in brief no. 195. Online.

Charcot-Marie-Tooth type I demyelinating neuropathies are genetically heterogeneous disorders (chrmosome 17,1,X). There are at least three genes on X chromosome, the more frequently involved being Cx32 in Xq13.1. Cx32 encodes for connexin-32, a gap junction protein of 283 aminoacids. We report the results of molecular studies in a CMTX1 Italian family, in which the mutation, found in the 5'-UTR, resulted in an abnormal mRNA connexin-32 expression. Mutations in PMP22 and P0 genes were also excluded in this family. Cx32 gene analysis carried out by PCR-SSCP on family members genomic DNAs, running a 321 bp fragment spanning the TATA box, the trasciptional start site, and the non coding exon 1b, revealed a shift correlated with a transition from C to T at position 40 of exon 1b of the 12 affected members, while was not found in the controls. Then the RT PCR-SSCP on cDNA from two peripheral nerve biopsies of two heterozygous females of the family were sequenced showing only the wild-type alleles and suggesting that mutated mRNAs were too unstable to be detected. The result also suggests a regulating role of the 5'-UTR of Cx32 mRNA.

5' Untranslated Regions↗
PubMed1998

A morphometric approach to the study of human ovarian organogenesis.

This study deals with the morphometric approach used to describe the quantitative characteristics of human ovaries during their final histogenesis. Fixed ovaries ranging from the 20th fetal week to the 8th postnatal month were used. They were measured using a high precision caliper, were embedded in toto in epoxide resin, and systematically cut (1 microm-thick sections) orthogonal to the major axis of the organ. The surface areas were automatically measured, at low magnification, and ovarian volume was calculated using the Cavalieri method. The volume fractions of ovarian medulla, primitive cortical tissue, primordial, primary, secondary and Graafian follicles, and stroma were calculated by differential point counting at higher magnification. At the same magnification, the position of the different follicle categories in the cortex was estimated as percentage distance of the follicle from the cortico-medullary boundary. The number in unit cortex volume of the oocyte nuclei in primordial and primary follicles were derived by diameter analysis according to the Schwartz-Saltykov method, and the total follicle number for each ovary was obtained. The diameter of nuclear, cellular and follicular profiles of primary and primordial follicles in sections was also measured. Ovarian volume was about 30 mm3 at 20 fetal weeks and 287 mm3 at the 8th postnatal month, with a 9.5-fold increment. Irrespective of age, the cortex occupied most of the organs, ranging from 90% at 20 prenatal weeks (about 27 mm3) to 85% at 8 postnatal months (about 245 mm3). In the cortex, the interstitium or stroma intermingled with the germinal component: primitive cortical tissue was found in all ovaries, and its absolute volume remained unmodified between the 20th fetal week and birth. In the analyzed fetal stages, the total number of primordial follicles had its largest modification, with a 1.3-fold increment between the 20th-25th fetal weeks. Hereafter, the number increased at a slower rate (about 1.2 times between the 25th week and birth, 1.1 times between birth and the 8th postnatal month). The largest absolute volume of primordial follicles was found at birth. Follicular, oocyte and nuclear diameters increased during the intrauterine life, while after birth their dimensions did not modify further. Conversely, their spatial position in the cortex modified even after birth. In the 8-month old child nearly all germinal cells were comprised into follicles in different degrees of development, and the primitive cortical tissue was exhausted. A three-dimensional geometrical model of human ovary has also been elaborated to define the spatial organization of ovarian germinal and somatic components, and to allow the comparison of the present data to the "in-vivo" volumetric assessments.

Embryonic and Fetal Development↗
PubMed1998

Analysis of fetal sex in TCC sample DNA: a contribution to the validation of this approach.

We compared two methods of collection of transcervical cell samples, mucus aspiration and cytobrush, with respect to the efficiency in determining fetal sex and we correlated the results with the week of gestation (7-11 weeks) to evaluate if the age of gestation influenced the success of the analysis. DNA extracted from TCC samples recovered by mucus aspiration (n = 27) and cytobrush (n = 36) were analysed by nested PCR to predict fetal sex. The statistical indices of sensitivity, specificity, positive predictive value and negative predictive value were determined, and compared with those of other studies previously performed. No statistically significant difference was found between the two methods of TCC sampling concerning the success of fetal sex prediction which was high for both methods (78 per cent and 89 per cent) and no correlation with the week of gestation was found. Transcervical cell sampling represents an encouraging prospect for first-trimester prenatal diagnosis even when the least invasive techniques are used.

Cervix Mucus↗
PubMed1997

Escape from X inactivation of two new genes associated with DXS6974E and DXS7020E.

Most genes on the X chromosome undergo "inactivation," being transcribed from only one copy in female somatic cells, but several human genes have been shown to be expressed from both the active and the otherwise inactivated homologue. To assess further the fraction and location of genes that escape inactivation, we have analyzed the inactivation status of a set of 73 expressed sequence tags that were derived from the sequencing of cDNA collections and mapped to the X chromosome. Of 33 that were expressed in cultured cells, as assessed by reverse transcription and PCR, 4 (about 12%) were transcribed from both the active and the inactive X chromosome. Two, RPS4 and PCTAIRE1, are already known to escape inactivation; the other 2, of unknown function, include a short cDNA with a full open reading frame and a transcript with no detectable open reading frame. They map, respectively, to Xp11.3-p11.4 and Xp22.2; both regions were previously reported to encode sequences transcribed from the inactive X. Neither transcript has a corresponding sequence on the Y. Thus, they exhibit double dosage in females compared to males, and inactivation status may be inconsequential for these transcribed sequences.

Base Sequence↗
PubMed1997

Mapping of 59 EST gene markers in 31 intervals spanning the human X chromosome.

The positioning of Expressed Sequence Tags (ESTs) constitutes an important step towards a functional map of the human genome, including candidate genes for human genetic disorders that have been localized by linkage analysis. We localized 59 ESTs on the human X chromosome, including 44 derived from infant brain and 15 from adult muscle cDNA libraries. Localizations by a somatic cell hybrid panel were refined for five cDNAs by mapping them in yeast artificial chromosome (YAC) contigs.

Animals↗
PubMed1997

Emx2 developmental expression in the primordia of the reproductive and excretory systems.

The development of the urogenital system has always attracted many investigators owing to the peculiar aspects of the embryology of the reproductive and excretory organs and to the high number of congenital anomalies related to these structures. It is remarkable because of the common origin of the kidneys, gonads, and genital tracts from the intermediate mesoderm and because differentiation of these organs involves extensive mesenchyme to epithelium transition. Our knowledge about the molecular mechanisms controlling the differentiation of these diverse structures from the same precursor has taken advantage of gene expression data and gene-targeting experiments using genes with a specific expression pattern in the urogenital system. A more detailed function in kidney development has been postulated for transcription factors such as WT-1, Pax-2 or other molecules such as glial cell line-derived neurotrophic factor (GDNF), Wnt-4, c-ret. In the present work we have described the expression pattern of the homeobox-containing gene Emx2 during the development of the urogenital system in mouse embryos. We have found that Emx2 is expressed in the early primordia of the organs that will form the excretory and reproductive systems. In particular we have found that Emx2 is expressed in the epithelial components of pronephros and mesonephros, in Wolffian and Müllerian ducts, in the ureteric buds with their branches and in the early epithelial structures derived from metanephrogenic mesenchyme. Emx2 is also intensely expressed in the "bipotential" or "indifferent" gonads and ovaries. These data and the recent finding that Emx2 homozygous mutant mice die soon after birth because of the absence of kidneys indicate an essential role of Emx2 in the morphogenesis of the urogenital system.

Animals↗
PubMed1997

Apoptosis of germ cells during human prenatal oogenesis.

During human oogenesis two contrasting processes can be observed: germ cell proliferation and differentiation, and contemporaneous germ cell death. It is well known that apoptosis is a type of physiological cell death that occurs in proliferating and differentiating tissues. The aim of this study is to demonstrate, through ultrastructural and in-situ 3' end labelling observations in intact sections of human fetal ovaries, that germ cell loss during fetal life is due to a phenomenon of apoptosis. We evaluated the presence of programmed cell death in female germ cells in fetal ovaries at 18-20 weeks of postconceptional age. The alterations that occur during apoptosis were detected by the electron microscope and include cytoplasmic condensation, organelle relocalization and compaction, chromatin condensation, and finally, production of membrane-enclosed particles containing intracellular material, known as apoptotic bodies, that are phagocytosed. The fragmentation of DNA, characteristic of apoptotic cells, was detected by the use of the in-situ 3' end labelling procedure on histological sections of ovaries where only some nuclei of germ cells were positively stained. The parallel use of these two methods on human ovaries of 18-20 weeks postconceptional age has allowed us to show that the numerical decrease of human female germ cells during the fetal period is due to an apoptotic phenomenon.

Apoptosis↗
PubMed1996

On the assessment of the growth patterns in human fetal limbs: longitudinal measurements and allometric analysis.

The total length (TL) and length of the ossified part (OL) of some long bones of the upper (humerus, ulna, radius) and lower limb (femur, tibia, fibula) were evaluated in 58 aborted human fetuses (crown-rump length, CRL, between 38 and 116 mm, developmental age from 8 to 14 weeks). The specimens, without any detectable malformation, were cleared and double-stained with alcian blue and alizarin red S to obtain a differential detection of the ossified part within the comprehensive outline between the cartilaginous epiphyses. The correlation between the values of TL and OL and those of CRL emphasized that the systematic OL measurement in limb long bones correlated better than TL with development age, since OL increased faster than TL. TL and OL also correlated with the CRL by bivariate allometry (ln y = ln a + b ln x) and the data obtained showed that they grew with positive allometry. The comparison between the cumulative values of the bones examined in each limb showed that both TL and OL grew relatively faster in the lower limb than the upper; the greatest growth rate was found for OL in the lower limb. These results many provide a tool for a comprehensive assessment of long bone growth patterns and may be useful in determining fetal growth even in incomplete specimens, in which one or some long bones can still be measured.

Embryonic and Fetal Development↗