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Cystic fibrosis (CF) is the main genetic cause of death among the Caucasian population. The disease is characterized by abnormal fluid and electrolyte mobility across secretory epithelia. The first manifestations occur within hours of birth (meconium ileus), later extending to other organs, generally affecting the respiratory tract. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR encodes a cyclic adenosine monophosphate (cAMP)-dependent, phosphorylation-regulated chloride channel required for transport of chloride and other ions through cell membranes. There are more than 2,000 mutations described in the CFTR gene, but one of them, phenylalanine residue at amino acid position 508 (p.F508del), a recessive allele, is responsible for the vast majority of CF cases worldwide. Here, we present the results of the application of genome-editing techniques to the restoration of CFTR activity in p.F508del patient-derived induced pluripotent stem cells (iPSCs). Gene-edited iPSCs were subsequently used to produce intestinal organoids on which the physiological activity of the restored gene was tested in forskolin-induced swelling tests. The seamless restoration of the p.F508del mutation resulted in normal expression of the mature CFTR glycoprotein, full recovery of CFTR activity, and a normal response of the repaired organoids to treatment with two approved CF therapies: VX-770 and VX-809., Graphical Abstract, Cystic Fibrosis (CF) is the main genetic cause of death among the Caucasian population. Here, we present the results of the application of genome-editing techniques to repair the CF defect in patient-derived cells and organoids. This protocol could be the foundation for future treatments based on cell repair and transplantation.

Background In countries where comparative genomic hybridization arrays (aCGH) and next generation sequencing are not widely available due to accessibility and economic constraints, conventional 400–500-band karyotyping is the first-line choice for the etiological diagnosis of patients with congenital malformations and intellectual disability. Conventional karyotype analysis can rule out chromosomal alterations greater than 10 Mb. However, some large structural abnormalities, such as derivative chromosomes, may go undetected when the analysis is performed at less than a 550-band resolution and the size and banding pattern of the interchanged segments are similar. Derivatives frequently originate from inter-chromosomal exchanges and sometimes are inherited from a parent who carries a reciprocal translocation. Case presentation We present two cases with derivative chromosomes involving a 9.1 Mb 5p deletion/14.8 Mb 10p duplication in the first patient and a 19.9 Mb 5p deletion/ 18.5 Mb 9p duplication in the second patient. These long chromosomal imbalances were ascertained by aCGH but not by conventional cytogenetics. Both patients presented with a deletion of the Cri du chat syndrome region and a duplication of another genomic region. Each patient had a unique clinical picture, and although they presented some features of Cri du chat syndrome, the phenotype did not conclusively point towards this diagnosis, although a chromosomopathy was suspected. Conclusions These cases highlight the fundamental role of the clinical suspicion in guiding the approach for the etiological diagnosis of patients. Molecular cytogenetics techniques, such as aCGH, should be considered when the clinician suspects the presence of a chromosomal imbalance in spite of a normal karyotype.

Background Hand-foot-genital syndrome (HFGS) is a rare condition characterized by congenital malformations in the limbs and genitourinary tract. Generally, this syndrome occurs due to point mutations that cause loss of function of the HOXA13 gene, which is located on 7p15; however, there are some patients with HFGS caused by interstitial deletions in this region. Case presentation We describe a pediatric Mexican patient who came to the Medical Genetics Department at the National Institute of Pediatrics because he presented with genital, hand and feet anomalies, facial dysmorphisms, and learning difficulties. Array CGH reported a 12.7 Mb deletion that includes HOXA13. Conclusions We compared our patient with cases of HFGS reported in the literature caused by a microdeletion; we found a minimum shared region in 7p15.2. By analyzing the phenotype in these patients, we suggest that microdeletions in this region should be investigated in all patients with clinical characteristics of HFGS who also present with dysplastic ears, mainly low-set implantation with a prominent antihelix, as well as a low nasal bridge and long philtrum.

ResumenIntroducciónLas eventraciones resultantes de incisiones de Kocher (transversa de Ross –subcostal bilateral– y transversal ampliada de West) condicionan la separación progresiva de los segmentos musculares de los rectos, con lo que su reparación resulta difícil, al quedar la sutura con una tensión excesiva cuando se consigue.En estas circunstancias se recurre a las prótesis para cubrir el defecto, interponiéndolas entre los extremos musculares y cerrando el defecto musculoaponeurótico sin tensión, evitando además la clínica de la eventración y eventuales complicaciones. No obstante, la solución ideal debería conseguir restituir la anatomía y función de los rectos, y que las prótesis queden como refuerzo de la autoplastia y no en sustitución del tejido autólogo.MétodoEntre marzo de 2012 y enero de 2015 operamos a 7 pacientes con eventraciones de una incisión de laparotomía transversa subcostal uni- o bilateral. Con el paciente en posición convencional, decúbito supino, en ningún caso era posible la sutura directa de los extremos de los rectos; la disección de los planos musculoaponeuróticos y la colocación del paciente en posición de semisentado en la mesa de quirófano (con la consiguiente aproximación de ambos extremos musculares) permitió la sutura sin una tensión excesiva.ResultadosEn todos los casos reparamos la continuidad de los músculos rectos con sutura directa. En el seguimiento de los pacientes, hasta la fecha, no se ha observado ningún caso de recidiva de la hernia incisional.ConclusiónLa reparación anatómica de la eventración subcostal, restableciendo la continuidad de la musculatura recta del abdomen, es posible en la mayoría de los casos y debería intentarse siempre.AbstractIntroductionThe incisional hernias resulting from Kocher incision, Ross incisión (bilateral subcostal) and the West transversal incision, they determine the progressive separation of the rectus muscle, resulting in a defect which repair it will be difficult to carry out because an excessive stress of the binding suture, when achieved.In these circumstances, it resorts to prosthesis to cover the defect between the edges of the rectus muscle. Thereby it is possible to close the musculoaponeurotic defect without tension, avoiding hernia clinic and its possible complications. However, the ideal solution should restore the anatomy and function of the muscles, with the prosthesis to reinforce the autoplasty, and not to replace the autologous tissue.MethodsBetween March 2012 and January 2015 we have operated seven patients with incisional hernias from transverse subcostal incision laparotomy. With the patient in the conventional position, supine, in neither case it was possible to suture the edges of the rectus muscles; after dissection of musculo-aponeurotic plane, patient was placed in semi-sitting position on the operating table, with consequent approach of both muscle suture edges thus allowing its suture without undue stress.ResultsIn all cases it was possible to repair the continuity of the rectus muscles with direct suture. In monitoring patients it has not been observed any relapse case of incisional hernia, to date.ConclusionThe anatomical repair of the subcostal incisional hernia, restoring the continuity of the rectus muscles of the abdomen, it is possible in most cases and should always be attempted.

Recently, 2q24.2 deletion syndrome has emerged as a cause ofintellectual disability. Takatsuki et al. [2010] provided the firstreportwithadeletionin2q24.2q24.3,confirmedusingcomparativegenomic hybridization microarray (aCGH) analysis, who had alow-birth weight, hypotonia, myoclonic seizures, dysmorphic fea-tures, and pulmonary emphysema. Subsequently, two other caseswere reported showing severe hypotonia, joint laxity, and facialdysmorphism;oneofthemhadadenovointerstitialdeletionof5-Mb at 2q24.2q24.3 [Magri et al., 2011], and the other patient (ID4547) who was included in the European Cytogeneticists Associa-tion Register of Unbalanced Chromosome Aberrations database,carrying a deletion in 2q24.2. The emerging syndrome was firstsuggestedbyPalumboetal.[2012],whodescribedacasewitha7.5-Mb de novo deletion in 2q24.1q24.2 encompassing 59 genes, asdetectedusingSNParray.Theclinicalfeaturesincludedgeneralizedhypotonia, marked bruxism, and bilaterally hip dislocation.In addition, two patients with mosaicism have been reported;the first (ID 1576) in Database of Chromosomal Imbalance andPhenotype in Humans using Ensembl Resources, is carrier of adouble2q24.2 deletion, one segmentof0.63-Mb (meanratio 1)and other of 0.17-Mb (mean ratio 0.41) inside the same region,this can be interpreted as a mosaic of monosomy and nullisomywhose molecular and clinical description is incomplete, thereforeit is not possible to include her in the present study. The secondmosaic,reportedbyBurrageetal.[2013]isamildlyaffectedpatientwithshortstatureandmosaicismwith61%ofcellscarryinga0.4-Mbdeletionon2q24.2,whichencompassesonlythreegenes.Theysuggested that TBR1was responsible for the intellectual disabilityobserved and that the short stature was associated with thePSMD14 gene.Wepresentapatient,whowasrecruitedinaprospectiveresearchprojecttodetectgenomicabnormalitiesthroughaCGHinpatientswithintellectualdisability;theresearchwasreviewedandapprovedby the ethics committee of the Instituto Nacional de Pediatri´a.The patient, a 7-year-old boy is the first child of healthy andnonconsanguineous parents. He was born after an uneventful

Angiogenesis, the sprouting of new blood vessels from pre-existing vessels, is a remarkable feature of tumours growth and metastasis. The in vivo inhibition of this receptor by cyclic peptides containing RGD sequence may be used to selectively suppress these disease1. Our research group has developed a new methodology2 for the evaluation of new antiangiogenic compounds, based in the genetic expression analysis using CGH array system. The RGD mimetics activity can be modified by the presence of ester-bond3, Therefore, we decided to prepare some depsipeptides analogous to the RGD-β-lactam compounds and evaluate their activity performing a genetic expression analysis. We also have evaluated the activity of open-chain compounds without RGD formal structure. The cyclic depsipeptides have demonstrated a very effective inhibitory activity. In the other hand the open-chain compounds have a surprising behavior, demonstrating a similar gene activation. This way, we call into question the essential need of RGD sequence to have an interaction between ligand and the receptor of the integrin4, 5. da Ressurreicao, A. S. M.; Vidu, A.; Civera M.; Belvisi L.; Potenza, D.; Manzoni, L.; Ongeri, S.; Gennari, C.; Piarulli, U.; Eur. 2009, 15, 12184. Aizpurua, J. M.; Ganboa, J. I.; Palomo, C.; Loinaz, I.; Oyarbide, J.; Fernandez, X.; Belentova, E.; Fratila, R. M.; Jimenez A.; Miranda, J. I.; Laso, A.; Avila, S.; Castrillo, J. L.; ChemBioChem, 2001, 11, 401. Cupido, T.; Spengler, J.; Ruiz-Rodriguez, J.; Adan, J.; Mitjans, F.; Puilats, J.; Albericio, F.; Chem. Int. Ed., 2010, 49, 2732. Xiong, J. P.; Stehle, T.; Zhang, R.; Joachimiak, A.; Frech, M.; Goodman, S. L.; Arnaout, M. A.; Science, 2002, 296, 151. Craig, D.; Gao, M.; Schulten, K.; Vogel, V.; Structure, 2004, 12, 2049.

In humans, a germline mutation (c.309C>G) in the TWIST1 oncogene may predispose to breast cancer and its expression has been associated with tumour progression and metastasis. In this study, the feline TWIST1 gene was screened for sequence variations in 37 neoplastic and eight hyperplastic mammary gland lesions from cats. In addition, mRNA levels were examined in 15 mammary tumours and three cases of mammary hyperplasia by quantitative real-time reverse-transcriptase PCR. Feline mammary carcinomas had significantly lower levels of expression of TWIST1 mRNA than benign mammary tumours. No variations were identified in the TWIST1 coding region in feline mammary tumours and the mutation described in humans was not detected. However, two germline variants in the TWIST1 gene intron were identified in four and three carcinomas, respectively: GQ167299:g.535delG and GQ167299:g.460C>T. There was no association between these sequence alterations and TWIST1 mRNA levels.

Angiogenesis is a fundamental step in the transition of solid tumors from a dormant state to a malign one. Many of the low-molecular-weight anti-angiogenic drug candidates mimic the short peptide epitope Arg-Gly-Asp (RGD), disrupting the extracellular matrix/integrin adhesion and, ultimately, leading to tumor cell apoptosis. In contrast to the detailed structural information available for the extracellular adhesion inhibition of endothelial cells through the recognition of integrins (typically aVb3) by RGD peptidomimetics, [2] most aspects of possible intracellular angiogenic gene regulation caused by peptidomimetics remain unexplored. Importantly, dysfunction of this signaling system is suspected to be behind the resistance phenomena developed in anti-angiogenic therapies. Inside the endothelial cell, dozens of proteins mediate or control the signaling pathways of angiogenesis after integrin activation, but only a couple of kinases (JNK, ERK) and transcription factors (NFkB, FoxO) are able to promote gene regulation (Figure 1). In addition, large environmental ligands, such as vascular endothelial growth factors (VEGFs) or peptide hormones, are required to elicit proangiogenic gene regulation. In this context, we set out to design alternative lowmolecular-weight RGD probes for interaction with aVb3 integrin and gene regulation in HUVECs. Ideally, these peptidomimetics should: 1) contain a minimal scaffold to prevent undesired scaffold/integrin interactions, 2) have a uniform and predictable scaffold conformation, 3) bear a maximum of recognition groups, including hydrophobic or hydrophilic ones, and 4) permit the deletion of selected residues from the RGD triad without global shape change. We selected five-membered and four-membered small cyclic peptides for development. The residual flexibility of these cyclopeptides can be further constrained by incorporating lactam bridges between neighboring amino acids to stabilize protein secondary structure motifs characterized by combinations of b-turns and/or g-turns. Several lactam pseudopeptides resulting from such an extension of Freidinger’s concept (Scheme 1; 1!2) have been explored with the goals of mimicking receptor-bound conformations of bioactive peptides and of providing pharmacophore information for nonpeptide drug design. However, despite its apparent simplicity, this design is not always reliable for the precise positioning of a maximum number of recognition groups around the pseudopeptide cyclic core. Because the interresidual lactam bridge created by modification of the side chain (R) shares recognition and constraint functions, the design of mimetic libraries becomes difficult and non-general, when synthetically achievable. An alternative way to constrain cyclic peptides is based on the incorporation of a d-amino acid and an N-alkyl-amino acid into the macrocycle, as illustrated by the remarkable aVb3 antagonist cilengitide (3, cyclo-[Arg-Gly-Asp-d-Phe-N(Me)Val]) developed by Kessler et al. Here we report an alternative, more versatile solution to the above design problem, by introducing a-amino-b-lactam scaffolds originating from aCH/NH proton mimicry (Scheme 2; 1! 4). This “b-lactam scaffold-assisted design” (b-LSAD) approach is based on the separation of recognition and constraint groups, and has previously proven efficient for promotion of unusually stable type-II (II’) b-turn peptidomimetics from extended open native peptides. Accordingly, the straight application of the b-LSAD principle to RGD cyclic mimetics led us to the b-lactam pseudopeptides 4a–d, which fulfilled several structural requirements to trigger quite different signaling interactions with aVb3 integrin. The hydrophobic benzyl group in the d-Phe residue of cilengitide (3), for instance, was replaced by the strongly hydrophilic 1,2-dihydroxyethyl moiety in the mimetic 4a or by the poorly hydrophobic methyl group in compound 4b. Conversely, the hydrophobic R–R substituents Figure 1. Signaling pathways initiated by integrins affecting the gene regulation of angiogenesis. ERK=extracellular-signal-regulated kinase, FAK= focal adhesion kinase, FoxO= forkhead box O, JNK=c-Jun N-terminal kinase, NFkB=nuclear factor k of B-cells.

Background The 15q11q13 region is subject to imprinting and is involved in various structural rearrangements. Less than 1% of Angelman Syndrome patients are due to translocations involving 15q11q13. These translocations can arise de novo or result from the segregation of chromosomes involved in a familial balanced translocation. Results A 5-year-old Mexican girl presented with developmental delay, minor dysmorphic features and history of exotropia. G-banding chromosome analysis established the diagnosis of Angelman Syndrome resulting from a familial translocation t(10;15) involving the 15q11.2 region. The available family members were studied using banding and molecular cytogenetic techniques, including Microarray-based Comparative Genomic Hybridization, which revealed additional unexpected results: a coincidental and smaller 15q deletion, asymptomatic duplications in 15q11.2 and Xp22.31 regions. Conclusions This report demonstrates the usefulness of array CGH for a detailed characterization of familial translocations, including the detection of submicroscopic copy number variations, which would otherwise be missed by karyotype analysis alone. Our report also expands two molecularly characterized rare patient cohorts: Angelman Syndrome patients due to familial translocations and patients with 15q11.2 duplications of paternal origin.

Infertility is an increasing problem all over the world, and it has been estimated that 10–15% of couples in fertile age have fertility problems. Likewise induced unsafe abortion is a serious threat to women’s health. Despite advances made in assisted reproduction techniques, little progress has been made in increasing the success rate during fertility treatment. This document describes a wide range of projects carried out to increase the understanding in the field of embryo implantation research. The ‘Fruitful’ research network was created to encourage collaborations within the consortium and to describe our different research potentials to granting agencies or private sponsors.

Here we report the identification and expression analysis of two novel human genes—DIPLA1 (Differentially expressed in placenta 1) and DIPAS (DIPLA1 Antisense). These genes are located at chromosomal region 9q33.1, in opposite orientations, and are flanked by the pregnancy-associated plasma protein-A (PAPP-A) and astrotactin 2 (ASTN2) genes. The mRNA sequences of both genes contain several upstream AUGs (uAUG) and various potential open reading frames (ORFs). DIPLA1 mRNA is 1.8 kb long and contains a 285 nt ORF coding for a polypeptide designated as replicative senescence up-regulated (RSU) protein. Antisense DIPAS mRNA is 2.7 kb long and contains a 309 nt ORF coding for a protein with partial similitude to the γ isoform variant of the human Ca2+/calmodulin (CaM)-dependent protein kinase II. Both genes are conserved in placental-species and are presumably transcribed from initiator (Inr) promoter elements located at opposite strands. In 20 human normal tissues tested, DIPLA1 mRNA expression was placenta-specific, whereas DIPAS mRNA expression was higher in placenta, brain, kidney and testis. In addition, DIPAS mRNA hybridizes with the 3′UTR region from PAPP-A mRNA, which spans over 4 kb more than previously reported, forming a potential sense-antisense double stranded RNA (dsRNA) duplex. Our results are of interest for placenta gene expression regulation and for the identification of novel genes in the human genome.

In this study, we performed the differential display technique to identify genes specifically expressed in human choriocarcinoma cell lines (JEG-3, JAR and BeWo) and normal placental term cells. Few differences were found among the expression profiles of the three choriocarcinoma cell lines and most of the differentially expressed genes were detected in normal term placenta. A total of 36 cDNA fragments were isolated and analysed. Of these, 19 sequences corresponded to regions in the human genome coding for potential novel genes. We confirmed by RT-PCR, the placental mRNA expression of three selected new human genes, on chromosomes 16q12, 9q32 and 6q22. The other 17 sequences showed high similarity to known human genes (like PSG3, FN1, PAI-2). Interestingly, the functions of five known proteins (from genes IK, TRA-1, HERPUD1, UBA-2, and TRAP240) have not yet been well characterized in placenta tissue. In addition, new alternative spliced mRNAs were detected for IK, TRAP240 and PLAC3 genes. The differential expression of the PAI-2 gene among the choriocarcinoma cell lines was also confirmed. The genes identified in this analysis will be of interest for future studies regarding both a better understanding of the biology of the trophoblast cell and the formation of placental tumors.

Anterior pituitary differentiation is a well-established paradigm in mammalian organogenesis. PIT1/GHF1, a homoeotic gene, plays a key role in terminal pituitary differentiation. Recently, a new set of transcription factors involved in early pituitary differentiation have been identified: Lhx-3, Lhx-4, P-OTX and Prop1. A pituitary-specific transcriptional cascade regulating a developmental programme leads to the determination of the mature cell types.

We report here the complete nucleotide sequence of a cDNA clone encoding Sparus aurata GHF-1/Pit-1 isolated from an expression library prepared from gilthead seabream pituitary gland poly(A)+ RNA. The cDNA sequence (saGHF-1/Pit-1) encodes a protein of 371 amino acids (aa) containing a POU domain (aa 194-343) and a transactivation, STA domain (aa 1-128). Northern blot hybridization of pituitary RNA detected a single 3.0 kb band and a rat GHF-1/Pit-1 antiserum was found to immunoreact with pituitary protein species of 42 kDa by Western blot analysis. When compared with mammalian GHF-1/Pit-1 aa sequence, the POU and STA domains of saGHF-1/Pit-1 protein show 83% and 48% aa identity, respectively. In spite of the low homology of the transactivation domain, saGHF-1/Pit-1 is able to activate the transcription of the human growth hormone promoter.

Many chromosomal deletions encompassing the 2q23.1 region have been described ranging from small deletions of 38 kb up to >19 Mb. Most phenotypic features of the 2q23.1 deletion syndrome are due to a MBD5 gene loss independent of the size of the deletion. Here, we describe a male patient harboring a novel interstitial deletion encompassing the 2q22.3 q23.3 chromosomal region. Array-CGH revealed a 7.1 Mb deletion causing haploinsufficiency of several genes including MBD5, ACVR2, KIF5C, and EPC2. This patient presents with additional findings to those already described in individuals who have deletions of MBD5 including toes absence of halluces, pure red cell aplasia, and intestinal aganglionosis. Interestingly, in the deleted region there are previously identified regulatory sequences which are located upstream to ZEB2, which is associated with Hirschsprung disease (HSCR). Several genes have been associated with pure red cell aplasia, but to our knowledge, this is the first time that 2q deletion is associated with this phenotype. These additional findings should be added to the list of manifestations associated with 2q deletion, and provide support for the hypothesis that this individual has a true contiguous gene deletion syndrome. © 2015 Wiley Periodicals, Inc.

The pituitary-specific transcription factor Pit-1/GHF-1 is a member of the POU domain family of regulatory proteins. It is involved in the commitment and expansion of the somatotropic cell lineage and activates the transcription of a set of anterior pituitary genes. We have cloned the human PIT1/GHF1 gene and characterized the regulatory mechanisms controlling its promoter activation and regulation. A minimal promoter region (-102 to +15) contains the cis-acting elements that confer to the human PIT1/GHF1 gene a high basal transcriptional activity, the tissue-specific expression, and the autoregulation by Pit-1/GHF-1 protein. The upstream promoter region contains a multiplicity of Pit-1/GHF-1 binding sites that do not show any synergistic interaction with the minimal promoter. The transcriptional activity is negatively regulated by Oct-1 and mediated by an octamer-binding site (OTF). In addition, we have also identified a 12-O-tetradecanoylphorbol-13-acetate-responsive element, which overlaps with a Pit-1/GHF-1 binding site. A mutually exclusive binding of the activator protein-1 (AP-1) and Pit-1/GHF-1 has been observed on this composite site, and AP-1 was shown to down-regulate PIT1/GHF1 transcription.

Growth hormone (GH) transcription factor 1 (GHF-1) or pituitary transcription factor 1 (Pit-1) is a pituitary gland-specific homeodomain protein of the POU subclass. POU proteins exert critical developmental and transcriptional functions. Two functional regions, the DNA-binding or POU domain and the transcriptional serine-threonine activation domain (STA) are highly conserved between fishes and primates. However, a variability in the length of the STA domain (between 71 and 128 amino acid residues) is found in different species as well as isoforms generated by alternative splicing.

The POU domain protein GHF-1 has a critical role in generation, proliferation and phenotypic expression of three pituitary cell types. GHF-1 functions in part by binding to and transactivating the promoters of both the growth hormone (GH) and prolactin (PRL) genes and that of the GHF1 gene itself. We describe a naturally occurring isoform of GHF-1, GHF-2, in which an additional 26 amino acids are inserted into the activation domain of the protein as a result of alternative splicing. GHF-2 retains the DNA binding activity of GHF-1 and can activate the GH promoter but has lost the ability to activate the PRL and GHF1 promoters. These results suggest that GHF-2 may function in differential target gene activation during differentiation of the somatotrophic lineage. Both GHF-1 and GHF-2 transcripts are specifically expressed in the anterior pituitary. Analysis of the genomic GHF1 gene shows that most of the distinct functional domains of GHF-1 (and GHF-2) are encoded by separate exons. Gene segment duplication and exon shuffling may have contributed to the evolution of this cell type-specific transcriptional regulatory gene.

The relationship between expression of the pituitary-specific transcription factor, GHF-1, and activation of the growth hormone and prolactin genes during mouse anterior pituitary development was investigated. While GHF-1 transcripts were detected within 24 hr of the first observable events in anterior pituitary differentiation, no GHF-1 protein could be detected until about 3 days later. The appearance of GHF-1 protein showed good temporal and spatial correlation with activation of the growth hormone gene. Prolactin gene expression, on the other hand, was observed transiently during embryonic day 16 in two different populations of cells, of which the major one does not contain GHF-1 or growth hormone. These results suggest that expression of GHF-1 is controlled both transcriptionally and posttranscriptionally. The spatial and temporal correlation between the appearance of GHF-1 protein and growth hormone gene activation suggests that GHF-1 is responsible for this very last step in the specialization of somatotrophic cells.

The growth hormone (GH) gene is specifically expressed within specialized cells of the anterior pituitary. The central role in GH gene activation is played by GHF-1, a homeodomain protein that is itself specifically expressed in the anterior pituitary.

Expression of POU1F1 gene, a member of the POU homeodomain family of transcription factors, is necessary for normal differentiation, development and survival of three anterior pituitary cell types (thyrotrophs, somatotrophs and lactotrophs) and for the proper expression of growth hormone (GH), prolactin (PRL), thyroid-stimulating hormone (TSH) genes and POU1F1 gene itself. Alternative splicing forms of this gene have been reported in different species, with few functional studies. Apart from the POU1F1-Wild-type with the expected length, in this work we isolated three additional splicing variants: POU1F1-beta, with a 78 bp insert in the trans-activation domain; POU1F1-gamma that lacks exon 3 and POU1F1-delta that lacks exons 3, 4 and 5. Four different protein isoforms were also detected by Western blot in the sheep pituitary tissue. Functional assays were performed to study the trans-activation of GH and PRL promoters by the splicing variants. Regarding the PRL promoter, the beta variant presented only 12% of the Wild-type trans-activation capacity. Variants gamma and delta showed no capacity to trans-activate PRL promoter. Both gamma and delta variants acted as repressors of Wt, reducing significantly the trans-activation made by Wt alone (p

POU1F1 (PIT-1/GHF-1) is a transcription factor with critical role in the transcriptional regulation of multiple genes in the pituitary and also important for the survival, differentiation and proliferation of three pituitary cell types. To understand the regulation of POU1F1 gene in Ovis aries we report its cloning, sequencing and characterization. The sequenced 5787 bp included six exons and two complete introns. Ovine POU1F1 gene has a high level of conservation with its bovine, human and rat counterparts showing 98.2%, 91.2% and 86.2% of similarity at the coding level, respectively. All six exons were analyzed for polymorphism detection in 100 animals of the Portuguese indigenous ovine breed 'Churra da Terra Quente'. One polymorphism was found at codon 58 in exon 2, in one allele of 4 animals leading to a change from cysteine to tyrosine (2% allelic frequency). In exon 3 two polymorphisms were detected: a G to A transition altering a glycine to an asparagine at codon 89 in one allele of one animal (0.5% allelic frequency) and another G to A transition at codon 105 converting an alanine into a threonine in one allele of 3 animals (1.5% allelic frequency). These polymorphisms might change the structure of the POU1F1 protein and modify gene-expression. In intron 4, an A to G transition was detected in one allele of six animals (3% allelic frequency). Exons 1, 4 and 6 showed no polymorphisms.

Here we report the selective expression of two POU transcription factor genes, PLA-1 and OCT-1, in human placenta and choriocarcinoma cell lines JAR, JEG-3 and BeWo. Pla-1 protein binds to a POU-consensus DNA sequence in the human placental lactogen-3 (PL-3) promoter and it is capable of trans-activating its transcription up to 18-fold. Other tissue-specific or ubiquitous POU transcription factors such as Pit-1/GHF-1 or Oct-1 showed none or low levels of trans-activation of the PL-3 promoter. In addition, we identified an unique and highly charged region in the N-terminal portion of Pla-1 protein required for full trans-activation of the PL-3 promoter.

In the past, human endometrial receptivity has been investigated by chasing specific molecules throughout the menstrual cycle. Now the genomic approach allows us to investigate the hierarchical contribution of a high number of genes to a specific function. In this study, we analyzed differentially the gene expression pattern of 375 human cytokines, chemokines, and related factors, plus that of their receptors, in endometrial receptivity. To do this, we used a combined approach of human endometrium and cell lines. We have compared the gene expression pattern in receptive vs. prereceptive human endometria and contrasted the results with gene expression in the highly adhesive cell line (to JAR cells and mouse blastocysts) RL95-2 vs. HEC-1A, a cell line with markedly less adhesiveness. IGF-binding protein-related protein 1 (IGFBP-rP1), also known as IGFBP-7/mac 25, was the second most up-regulated gene in both of the investigated models. These results were corroborated by performing RT-PCR on the same RNA samples and validated by quantitative fluorescent RT-PCR and in situ hybridization in endometrium throughout the menstrual cycle. Interestingly, a 35-fold increase in expression during the receptive phase was compared with the prereceptive phase followed by a sharp increase in the late luteal. Further quantitative fluorescent RT-PCR experiments using the epithelial and stromal endometrial fraction throughout the menstrual cycle confirmed that IGFBP-rP1 expression was localized in the epithelial and stromal compartments and up-regulated mainly in the latter. In situ experiments confirmed the endometrial localization and regulation of IGFBP-rP1 mRNA. At the protein level, IGFBP-rP1 was localized by immunohistochemistry at the apical part of the luminal and glandular epithelium, stromal, and endothelial cells. In conclusion, using a genomic approach with a combined experimental design of receptivity in vivo and in vitro, we have discovered the implication of IGFBP-rP1 in endometrial physiology, which seems related to endometrial receptivity.

Endometrial receptivity is a self-limited period in which the endometrial epithelium (EE) acquires a functional and transient ovarian steroid-dependent status that allows blastocyst adhesion. Termed as "the window of implantation", this specific period opens 4-5 days after progesterone production or administration and closes after 9-10 days. Scientific knowledge on the endometrial receptivity process is fundamental for the understanding of human reproduction, but so far none of the proposed biochemical markers for endometrial receptivity has been proven to be clinically useful. In this work, we present strategies of cDNA analysis technologies that aim to clarify the fragmented information in this field. Specifically, the objective is the differential identification, cloning and sequencing of genes linked to endometrial receptivity in humans, combining differential display PCR and cDNA microarray analysis of endometrial epithelial-derived cell lines and endometrial samples obtained in the same patient 2 and 7 days after the luteinizing hormone (LH) surge (day LH+2) and (day LH+7), respectively.

The rat growth hormone (GH) promoter was significantly activated in non-pituitary cells by the expression of unliganded trioodothyronine (T3) and retinoic acid (RA) receptors. Furthermore, a strong ligand-dependent activation was found in the presence of the pituitary-specific transcription factor GHF-1. When compared with GHF-1, the splice variant GHF-2 showed a decreased ability to bind the cognate site in the GH promoter. As a consequence, expression of GHF-2 had little stimulatory effect on the GH promoter and did not show cooperation with T3 or RA receptors even in the presence of ligands. Furthermore, over-expression of GHF-2 inhibited the response to T3 and RA in pituitary cells. These results show that alternative splicing of the GHF-1 gene gives rise to two isoforms that differ in their transactivating properties and in their ability to synergize with the nuclear thyroid hormone and retinoic acid receptors an GH gene expression., This work has been supported by the grants PB94–0094 and PB94–0047 from the DGICYT, BIO2-CT93–0473 from the EEC and from the Comunidad de Madrid.

We describe the expression of the transcription factor GHF-1/PIT-1 in adult porcine adenohypophysis by a nonradioactive in situ hybridization (ISH) method using a digoxigenin-labeled cDNA probe corresponding to the entire coding region of rat GHF-1. GHF-1 transcripts were found in 71.7% of adenohypophyseal cells. We also report the simultaneous detection of GHF-1 mRNA and pituitary hormones by combined ISH and immunocytochemistry (IC) in dispersed adenohypophyseal cells, detected with an alkaline phosphatase-NBT/BCIP technique and with an immunoperoxidase-3-amino-9-ethylcarbazole (AEC) method, respectively. The combination of the two techniques neither abolished nor diminished their sensitivity or specificity. GHF-1 is expressed in all five of the cell types in the adult porcine adenohypophysis, showing that this method is suitable for simultaneous detection of transcripts and proteins at the single-cell level.

The role of the POU domain transcription factor Pit-1 (GHF-1) in differentiation and proliferation of the somatotrophic lineage is well known. In our study of differential splicing of the PIT1 gene we found a new protein, Pit-2, in which 26 amino acids are inserted into the transactivation domain. Pit-2 can activate the growth hormone promoter but has lost the ability to activate the prolactin promoter or PIT1 promoter itself.

Mutations that cause pituitary dwarfism in the mouse reside in the gene encoding the transcription factor growth hormone factor 1 (GHF1 or pit1). These dwarf mice (dw and dwJ) are deficient in growth hormone (GH) and prolactin (PRL) synthesis and exhibit pituitary hypoplasia, suggesting a stem cell defect. With antisense oligonucleotide technology, a cell culture model of this genetic defect was developed. Specific inhibition of GHF1 synthesis by complementary oligonucleotides led to a marked decrease in GH and PRL expression and to a marked decrease in proliferation of somatotrophic cell lines. These results provide direct evidence that the homeodomain protein GHF1 is required not only for the establishment and maintenance of the differentiated phenotype but for cell proliferation as well.

Introduction: Chitotriosidase protein (CT) is the most important biochemical marker described for Gaucher disease (GD). CT activity is increased several hundred-fold in plasma of GD patients and shows a strong positive correlation with the severity of the disease. However, a recessively inherited enzyme deficiency, with an incidence of about 6% in the Caucasian population, means that not all patients with GD can be monitored by measuring CT activity. Materials and Methods: This study was performed with plasma samples from 22 type 1 GD patients and 23 healthy subjects. All patients are from Spanish GD Registry. Applying two dimensional gel electrophoresis (2-DE) we compared the plasma proteome profile of GD patients with that of healthy subjects. Differential proteins were excised and prepared for MALDI-TOF analysis. Results were verify with 2-DE western blot. Results: In this study we have described five CT isoforms in a 2-DE gel from GD plasma samples. We show and compare the 2-DE images of each CT spot in plasma protein samples from healthy controls, GD patients with wild-type CT protein, and GD patients either homozygous or heterozygous for the 24 bp duplication. We propose a connection between the quantitative image analysis of each CT spot and the enzymatic activity of CT in each patient. Lastly, our results verifies that post-translational glycosylation explains at least some of differences between CT isoforms. Conclusion: The possibilities of proteomic technology in the investigation of biological biomarkers of GD and the nature of the CT protein are demonstrated, as well as in detecting new biochemical markers useful for the diagnosis of the disease and for monitoring patient response to therapy.

THE specific expression of growth hormone (GH) in the somatotrophic cells of the anterior pituitary is largely attributable to a short promoter in the 5' flanking region of the GH gene1–6. This promoter contains two binding sites for the transcription factor GHF-1 (refs 3,7), the expression of which is also specific to cells of the somatotrophic lineage8,9 and correlates with activation of the GH gene in the developing mouse pituitary9. Various studies indicate that GHF-1 is the main determinant of cell type-specific expression of the GH gene3–9. GHF-1 is a member of the POU-domain class of proteins that each contain two highly conserved sequence motifs, the homoeodomain and the POU-specific domain8,10,11. Here we report that the GHF-1 homoeodomain is sufficient for sequence-specific DNA binding, although its activity is stimulated by the POU-specific domain, which does not interact directly with the DNA. Transcriptional activation is mediated by a separate domain rich in hydroxylated amino-acid residues. Similar sequences are present in other cell type-specific transcription factors.

Growth hormone (GH) expression in pituitary-derived cells has been attributed to the presence of a positive trans -activator, GHF-1, which binds to two sites on the GH promoter. Somatic cell hybridization of non-GH-expressing L cells with pituitary-derived GH3 cells usually results in extinction of GH production. While previous studies showed that extinction occurs at the level of GH transcription, the exact mechanism remained elusive. We therefore characterized two parental cell lines and three hybrids, two of which extinguish GH expression and one in which GH is reexpressed after loss of mouse chromosomal material. Using in vivo transfections, in vitro transcription, DNAase I footprints, and immunoblotting experiments, no evidence for a direct repressor of GH transcription was found. Rather, extinction of GH expression in fibroblast ⇔mes; pituitary hybrids was accompanied by loss of GHF-1 protein and mRNA expression, suggesting that extinction occurs by repression of this trans -activator.

HeLa cells infected with poliovirus exclusively synthesized proteins coded by the P2 and P3 regions of the viral genome when they were placed in an alkaline medium lacking sodium ions. The amount of viral protein synthesized was augmented by increasing the concentration of KCl. Also in the presence of KCl, the cells continued synthesizing this altered pattern of proteins for longer times. This effect occurred in the presence of guanidine, it was reversible, and the normal pattern of poliovirus proteins reappeared when control medium was added, even when guanidine was present. These findings suggest that truncated viral RNA is not made under these conditions. Pactamycin and sodium fluoride, two known inhibitors of the initiation of translation, blocked protein synthesis in cells placed in alkaline medium, indicating the possibility that initiation at internal sites of the viral mRNA may take place under these conditions. Finally, the proteins synthesized were analysed by two-dimensional gel electrophoresis. The proteins migrated as authentic viral proteins indicating that if internal initiation takes place, at least some of these initiation events occur in phase with the initiation codon present in the poliovirus genome.

Pituitary-specific expression of the growth hormone (GH) gene is governed by a transcription factor, GHF-1, that binds to two sites within its promoter. Recently, GHF-1 was shown to be a member of the homeobox family of DNA-binding proteins. An important question is whether GHF-1 controls the expression of other pituitary specific genes, such as prolactin (Prl), expressed in closely related cell types. To this end, GHF-1 was purified from extracts of GH- and Prl-expressing pituitary tumor cells and identified as a 33-kilodalton polypeptide. Although GHF-1 bound to and activated the GH promoter, it did not recognize the Prl promoter. However, at least one other factor in the same extracts, which was easily separated from GHF-1, bound to several sites within the Prl but not the GH promoter. Antibodies to GHF-1 did not react with the Prl binding activity. These results imply that the pituitary-specific expression of GH and Prl is governed by two distinct trans-acting factors.

The transcription inhibitor α-amanitin which does not easily pass through the membrane of normal cells, shows a higher inhibition of cellular RNA synthesis in encephalomyocarditis (EMC) virus-infected HeLa cells as compared with uninfected cells. This selective inhibition is observed within minutes of virus addition to the culture medium. It depends on the multiplicity of infection used and the concentration of α-amanitin present. These results indicate that, very early during infection, α-amanitin is able to pass the cell membrane and get into the nucleus, where it blocks RNA synthesis. No effect is observed in the transport of [3H]uridine into the cells, indicating that the increased inhibition by α-amanitin affects RNA synthesis itself. This phenomenon occurs with different animal viruses irrespective of whether they possess a lipid envelope or not. Finally, no inhibition by NH4Cl or chloroquine is observed in the permeabilization induced by encephalomyocarditis virus, indicating that it is not a low-pH requiring process. However, it does require a metabolically active cell, since NaN3 plus 2′-deoxy-glucose blocked this process.

Modification of membrane permeability has been analyzed in Semliki Forest virus (SFV)-infected cells by means of translation inhibitors not permeable to normal cells. A higher inhibition of protein synthesis in the infected cells is only observed with those antibiotics that do not easily pass the cell membrane, but not with others, permeable to cells, such as anisomycin, cycloheximide, trichodermin, etc. It does not, therefore, seem that the suggestion of M. A. Gray, K. J. Micklem, and C. A. Pasternak [Eur. J. Biochem.135, 299–302, (1983)] that protein synthesis in virus-infected cells is more susceptible to translation inhibitors in general, is correct. Both low- and high-molecular weight compounds enter the cell very early during SFV infection. This permeabilization is blocked by compounds known to increase the pH of coated vesicles, such as NH4Cl and chloroquine. Inhibition of energy production by means of N3Na and 2′-deoxyglucose also blocks this process. The optimal external pH for this early permeabilization is around 7–8. Acidic pH inhibits the entry of these impermeant antibiotics promoted by SFV. Analysis of 86Rb+ content in SFV-infected HeLa cells also indicates that a drastic decline in this cation takes place, in agreement with previous findings, but disagreeing with the previous results. A parallel between the decrease in this cation and the blockade of protein synthesis is apparent, throughout the course of infection. In addition to the early permeabilization that takes place during virus entry, increased entry of hygromycin B and a-sarcin also occurs in SFV-infected cells from 2 to 3 hr postinfection, but not when late viral replication is blocked by means of interferon treatment.

Poliovirus-infected HeLa cells increased their permeability to monovalent ions from the third hour post-infection. At that time infected cells lost their content of potassium ions as measured by efflux of the potassium analogue 86Rb+. The rate of release of 86Rb+ from cells increased as infection proceeded, and was not sensitive to ouabain or quinidine, inhibitors of the Na+/K+ ATPase and Ca2+-induced K+ release system, respectively. The leakage of 86Rb+ was only slightly sensitive to furosemide, an inhibitor of the Na+/K+/Cl− cotransport system, suggesting that the mechanism of release involved an increased passive permeability of the cell membrane. The calcium content or its efflux did not vary significantly in the infected cells. Neither were there alterations in the intracellular pH throughout infection., Ms M. A. Ramos is acknowledged for excellent technical assistance. A.L.R. and J.L.C. are fellows of the Ministerio de Education y Ciencia. CAICYT and FIS are acknowledged for financial support.

The effect of pH and different concentrations of added monovalent and divalent cations on translation in poliovirus-infected HeLa cells has been examined. A strong effect on protein synthesis was observed when the concentration of sodium ions was modified. If cells were placed in a hypotonic medium after virus adsorption, no shut-off of cellular translation took place, nor were viral proteins synthesized. An increase in the multiplicity of infection partially overcame this effect. Reversal of the shut-off of cellular translation and inhibition of viral protein synthesis was achieved when cells were placed in hypotonic medium 2 h after infection. Modification of divalent cations (calcium or magnesium) had little or no effect on the pattern of translation. On the other hand, acidic pH (below 6) inhibited both cellular and viral protein synthesis, whereas basic pH had no influence. During infection the synthesis of poliovirus proteins reached a maximum at about 4 to 6 h and then declined. This inhibition of viral translation was partially prevented if cells were placed in a medium containing a high concentration of potassium although the cytopathic effect was prevented. These results indicate that viral protein synthesis and the cytopathic effect were, to a large extent, influenced by the external monovalent ion concentration., The excellent technical assistance of Ms M. A. Ramos is acknowledged. J.L.C. and A.L.R. are holders of Ministerio de Educación y Ciencia fellowships. FIS and CAYCIT are acknowledged for financial support.

Picornavirus infection induces a profound inhibition of labelling of newly synthesized RNA in some cell lines. EMC virus blocks transcription in L929 cells, particularly at early times during infection. This inhibition is not dependent on virus gene expression, since it occurs with UV-inactivated virus and also in the presence of translation inhibitors. The inhibition can be largely accounted for by the blockade of [3H]nucleoside transport, as suggested by the transport kinetics and incorporation of labelled nucleoside from preloaded cells. The inhibition of transport and incorporation into TCA-precipitable material was observed with pyrimidine (uridine, thymidine and cytosine) and purine nucleosides (adenosine and guanosine), but the blockade by EMC virus was higher with the latter nucleosides. Preloading of cells with any of these nucleosides resulted in a decreased effect on nucleoside incorporation into nucleic acid after virus infection. These results suggest that the inhibition of incorporation of labelled nucleosides into nucleic acid in EMC virus-infected cells can be explained, at least in part, by the decreased pool size of the phosphorylated nucleosides. These effects are not specific for L cells, because they are also observed in other cell lines.

The growth hormone (GH) gene is uniquely expressed in specialized cells, known as somatotropes, in the anterior pituitary. Previous analysis has shown that the difference between GH gene activity in somatotropes and hepatocytes exceeds 108-fold (Ivarie et al, 1983). To achieve this level of control, a highly specific mechanism should restrict GH expression to the correct cell type. The GH-expressing somatotrope is derived from an acidophylic stem cell that appears during the ontogeny of the anterior pituitary which also gives rise to the prolactin (PRL)-expressing lactotrope (Chatelain et al., 1979). It is generally accepted that during development and differentiation of the rat pituitary, the acidophylic stem cell first gives rise to somatotropes (GH+, PRL−), which in turn give rise to additional somatotropes and to mammosomatotropes (GH+, PRL+). These latter cells are the major precursors of lactotropes (GH−, PRL+), the last cell type to appear along this lineage (Hoeffler et al., 1985; Behringer et al., 1988). Various data indicate the existence of a subpopulation of GH+ cells which can switch to PRL+ cells and PRL+ cells which can switch to GH+ cells, under the influence of certain hormones such as estrogens and GH-releasing factor (Leong et al., 1985; Strattmann et al., 1974). The regulation of cell type differentiation in this progressive lineage offers an interesting experimental system for studying the control of cellular differentiation.

Here we report the characterization of 12 kb genomic DNA upstream of the human PIT1/GHF1 promoter. Different regions involved in the modulation of human PIT1/GHF1 gene expression were defined by transient transfection studies. Two regions, one proximal (-7.1/-2.3) and one distal (-11.8/-10.9), presented an enhancer activity in pituitary cells when placed upstream of the SV40 promoter. The 0.9 kb distal region was analysed further and found to decrease the basal transcriptional activity of the human PIT1/GHF1 minimal promoter, indicating that this region behaves as a silencer for its own promoter. Three Pit-1/GHF-1-binding sites and two ubiquitous nuclear factor 1 (NF-1)-binding sites were identified by DNase I footprinting in the distal regulatory region. Deletion analysis indicated that NF-1 or NF-1-related protein(s) participate in the down-regulation of human PIT1/GHF1 gene expression by interacting with an NF-1-binding site within the distal regulatory region. The nucleotide sequence data reported will appear in DDBJ, EMBL and GenBank Nucleotide Sequence Databases under the accession number X97489.