Jorge Bolivar

Epigenetic silencers and Notch collaborate to promote malignant tumours by Rb silencing

Cancer is both a genetic and an epigenetic disease. Inactivation of tumour-suppressor genes by epigenetic changes is frequently observed in human cancers, particularly as a result of the modifications of histones and DNA methylation. It is therefore important to understand how these damaging changes might come about. By studying tumorigenesis in the Drosophila eye, here we identify two Polycomb group epigenetic silencers, Pipsqueak and Lola, that participate in this process. When coupled with overexpression of Delta, deregulation of the expression of Pipsqueak and Lola induces the formation of metastatic tumours. This phenotype depends on the histone-modifying enzymes Rpd3 (a histone deacetylase), Su(var)3-9 and E(z), as well as on the chromodomain protein Polycomb. Expression of the gene Retinoblastoma-family protein (Rbf ) is downregulated in these tumours and, indeed, this downregulation is associated with DNA hypermethylation. Together, these results establish a mechanism that links the Notch–Delta pathway, epigenetic silencing pathways and cell-cycle control in the process of tumorigenesis.

Genetic dissection of a stem cell niche: the case of the Drosophila ovary.

In this work, we demonstrate a powerful new tool for the manipulation of the stromal component of a well‐established Drosophila stem cell niche. We have generated a bric‐a‐brac 1 (bab1)‐Gal4 line that drives UAS expression in many somatic ovary cell types from early larval stages. Using this Gal4 line, we could effectively induce FLP/FRT‐mediated recombination in the stromal cells of the ovarian germline stem cell niche. Mutant clones were observed in the developing ovary of larvae and pupae, including in somatic cell types that do not divide in the adult, such as the cap cells and the terminal filament cells. Exploiting the ability of bab1‐Gal4 to generate large clones, we demonstrate that bab1‐Gal4 is an effective tool for analyzing stem cell niche morphogenesis and cyst formation in the germarium. We have identified a novel requirement for engrailed in the correct organization of the terminal filaments. We also demonstrate an involvement for integrins in cyst formation and follicle cell encapsulation. Finally using bab1‐Gal4 in conjunction with the Gal80 system, we show that while ectopic dpp expression from stromal cells is sufficient to induce hyperplastic stem cell growth, neither activation nor inactivation of the BMP pathway within stromal cells affects germline stem cell maintenance. Developmental Dynamics 235:2969–2979, 2006.

A brain circuit that synchronizes growth and maturation revealed through Dilp8 binding to Lgr3

Brain keeps body size and shape in check Animal systems show amazing left-right symmetry—think of how our legs or arms, or the legs or wings of an insect, are matched in size and shape. Environmental insults and growth defects can challenge these developmental programs. In order to limit the resultant variation, juvenile organisms buffer variability through homeostatic mechanisms, so that the correct final size is attained. Vallejo et al. report that the Drosophila brain mediates such homeostatic control via an insulin-like peptide Dilp8 binding to the relaxin hormone receptor Lgr3. Lgr3 neurons distribute this information to other neuronal populations to adjust the hormones ecdysone, insulin, and juvenile hormone in a manner that stabilizes body and organ size. Science, this issue p. 10.1126/science.aac6767 Drosophila Lgr3 defines a neural circuit for homeostatic regulation of body size. INTRODUCTION Animals have a remarkable capacity to maintain a constant size, even in the face of genetic and environmental perturbations. Size imperfections and asymmetries have an effect on fitness, potentially decreasing competitiveness, survival, and reproductive success. Therefore, immature animals must employ homeostatic mechanisms to counteract substantial size variations and withstand developmental growth perturbations caused by genetic errors, disease, environmental factors, or injury. Such mechanisms ensure that, despite inevitable variations, the appropriate final body size is attained. A better understanding of homeostatic size maintenance will afford insights into normal organ and organismal size control, as well as the developmental origin of anomalous random left-right asymmetries. RATIONALE The Drosophila insulin-like peptide Dilp8 has been shown to mediate homeostatic regulation. When growth is disturbed, Dilp8 is strongly activated and sexual maturation is postponed until the affected elements are recomposed; simultaneously, the growth of other organs is retarded during this process. This compensatory mechanism allows the growth of the affected tissues to catch up. It maintains the synchrony between organs so that the animals achieve the correct size, preserving proportionality and bilateral symmetry. However, the Dilp8 receptor and its site of action remain uncharacterized. RESULTS We found that Dilp8 binds to andactivates the relaxin leucine-rich repeat–containing G protein–coupled receptor Lgr3 to mediate homeostatic control through a pathway dependent on adenosine 3′,5′-monophosphate. Larvae that lack lgr3 in neurons alone do not respond to Dilp8, indicating that the homeostatic system is centered in the brain. Dilp8 delays reproductive maturation by suppressing the neurons releasing the prothoracicotropic hormone (PTTH), which projects to the prothoracic gland and regulates ecdysone production for growth termination. However, this modulation alone is insufficient to adjust growth and stabilize body size. We show that Dilp8-Lgr3 balances growth against the extended growth period by dampening the production of dilp3 and dilp5 by insulin-producing cells (IPCs) in the brain and inhibiting synthesis of the juvenile hormone (JH). We also identify two pairs of dorsomedial neurons in the pars intercerebralis that are necessary and sufficient to mediate the effects of Dilp8. Simultaneous detection of pre- and postsynaptic markers revealed that the Lgr3 neurons mediating this homeostatic control have extensive axonal arborizations. Genetic and GRASP (GFP reconstitution across synaptic partners) analyses demonstrate that these neurons are connected to both the IPCs and PTTH neurons critical for adjusting growth and maturation rate, respectively. Thus, through their extensive axonal arborizations, Lgr3 neurons function like a “neuronal hub”: They route peripheral information about growth status to other neuronal populations, thereby synchronizing damaged tissues and other (undamaged) ones and allocating additional development time so that each organ attains the correct size and maintains proportionality and symmetry. CONCLUSION We identified the relaxin receptor Lgr3 as a Dilp8 receptor and defined a brain circuit for homeostatic control of organismal and organ size in the face of perturbations. Lgr3 neurons that respond to Dilp8 signals directly input on the insulin-producing cells and the PTTH-producing neurons. As Lgr3 outputs, the modulation of these neuronal populations according to Dilp8 levels is critical to delay maturation and promote growth compensation in a manner that stabilizes body size. Without adequate Dilp8-Lgr3 signaling, the brain is incapable of stabilizing size between the distinct body parts, and we see left-right asymmetries and size variations that are greater than usual, reflecting developmental instability. Dilp8-Lgr3 neural circuit and outputs for body-size homeostasis. The brain detects growth status and anomalies via Dilp8 activation of the Lgr3 receptor in a pair of symmetric neurons. These neurons distribute this information to IPCs and PTTH neurons, which then trigger the hormonal responses that regulate size. Without Dilp8-Lgr3 homeostasis, the brain cannot correct variation, and identical body parts can display imperfect symmetry and size. Body-size constancy and symmetry are signs of developmental stability. Yet, it is unclear exactly how developing animals buffer size variation. Drosophila insulin-like peptide Dilp8 is responsive to growth perturbations and controls homeostatic mechanisms that coordinately adjust growth and maturation to maintain size within the normal range. Here we show that Lgr3 is a Dilp8 receptor. Through the use of functional and adenosine 3′,5′-monophosphate assays, we defined a pair of Lgr3 neurons that mediate homeostatic regulation. These neurons have extensive axonal arborizations, and genetic and green fluorescent protein reconstitution across synaptic partners show that these neurons connect with the insulin-producing cells and prothoracicotropic hormone–producing neurons to attenuate growth and maturation. This previously unrecognized circuit suggests how growth and maturation rate are matched and co-regulated according to Dilp8 signals to stabilize organismal size.

An inducible nitric oxide synthase (NOS) is expressed in hemocytes of the spiny lobster Panulirus argus: cloning, characterization and expression analysis.

Nitric oxide (NO) is a free radical gas involved in a variety of physiological processes in invertebrates, such as neuromodulation, muscle contraction and host defense. Surprisingly, little is known about the involvement of NO synthase (NOS) in the immune system of crustaceans. This work is focused on the study of the NOS gene of the spiny lobster Panulirus argus, a crustacean with commercial interest, and its relationship with the immune response to a microbial elicitor. A NOS full-length DNA was isolated from hemocytes by reverse transcription-polymerase chain reaction (RT-PCR) using degenerated primers. The open reading frame (ORF) encodes a protein of 1200 amino acids, with an estimated molecular mass of 135.9 kDa, which contains the conserved domains and binding motifs of NOS found in a variety of organisms. NOS gene expression in lobster gills, heart, stomach, digestive gland, abdominal muscle, gut and hemocytes was studied by Real Time quantitative PCR (Real Time qPCR). The expression was higher in hemocytes, heart and gills. In addition, when lobster hemocytes were exposed in vitro to Escherichia coli O55:B5 lipopolysaccharide (LPS), an increase in the NOS activity and also in the NOS gene expression evaluated by Real Time qPCR was observed, thus demonstrating the presence of an inducible crustacean NOS by a microbial elicitor of the immune response. The information is relevant in providing basic knowledge for further studies of crustacean defense mechanisms.

Molecular Cloning of a Zinc Finger Autoantigen Transiently Associated with Interphase Nucleolus and Mitotic Centromeres and Midbodies ORTHOLOGOUS PROTEINS WITH NINE CXXC MOTIFS HIGHLY CONSERVED FROM NEMATODES TO HUMANS

We have cloned a novel human autoimmune antigen in a patient suffering from rheumatoid arthritis with high levels of antibodies to the nucleolus organizer regions. Initially the human autoimmune serum was used to select a cDNA of 317 amino acids from a hamster expression library. Using the hamster DNA as a probe, we isolated the human homologous cDNA of 320 amino acids. Human and hamster polypeptides share a 95% amino acid homology. The deduced 36-kDa protein contains a putative amino-terminal NLS signal, nine cysteine-X-X-cysteine motifs highly conserved, and a carboxyl-terminal poly acidic region. Several homologous expressed sequence tags have been identified in data bases suggesting that orthologous proteins are present throughout evolution from worms to humans. A Drosophila expressed sequence tag was further completely sequenced for a full-length protein with 60% amino acid identity to the human homologue. Northern blot analysis revealed that this novel protein is widely distributed in human tissues with significantly higher expression levels in heart and skeletal muscle. Specific antibodies to the recombinant protein and transfection experiments demonstrated by immunofluorescence the localization of the protein predominantly but not exclusively to the nucleolus of interphase mammalian cells. In actinomycin D-treated cells the protein remains associated with the nucleolus but is not segregated, like other ribosomal factors such as upstream binding factor. In mitosis the protein was found to be associated with centromeres and concentrated at the midbody in cytokinesis. Transient distribution of this evolutionarily conserved zinc finger nucleolar autoantigen to the mitotic centromeres may provide the means for several aspects of cell cycle control and transcriptional regulation.

Immunohistochemical Detection of Ribosomal Transcription Factor UBF and AgNOR Staining Identify Apoptotic Events in Neoplastic Cells of Hodgkin's Disease and in Other Lymphoid Cells

Ribosomal RNA synthesis is a key molecular process for understanding the mechanisms that drive cell proliferation. In this process, the upstream binding factor (UBF) is involved in regulating rDNA transcription at the nucleolus, together with RNA polymerase I. Recently, UBF was demonstrated to be a substrate for selective cleavage by specific proteases during apoptosis. Here we studied the expression of UBF in several cases of Hodgkins disease (HD) by immunostaining and found it to be absent or clearly diminished in a high proportion of Reed–Sternberg cells and Hodgkin cells compared to small reactive lymphocytes. This result contrasted with labeling of those cells by the AgNOR technique, a marker of cell proliferation dependent on increased amounts of several proteins related to ribosome assembly. Disappearance of UBF and preservation of other NOR proteins is consistent with the pattern of selective proteolysis by caspases described in early stages of apoptosis. This correlates well with our results observed on induction of apoptosis in Jurkat cells treated with anti-FAS/APO-1 serum and with those in aged germinal center B-cells, in which UBF was no longer seen although the staining signal of other NOR proteins was maintained. These results support the concept that the rate of apoptosis is higher in neoplastic cells of HD than in the benign reactive lymphocyte population. Differential proteolysis of NOR proteins, as revealed by double staining of UBF and AgNOR, may prove valuable for identification of early stages of apoptosis in cytological and histopathological samples.

Identification of enhanced hydrogen and ethanol Escherichia coli producer strains in a glycerol-based medium by screening in single-knock out mutant collections.

BackgroundEarth’s climate is warming as a result of anthropogenic emissions of greenhouse gases from fossil fuel combustion. Bioenergy, which includes biodiesel, biohydrogen and bioethanol, has emerged as a sustainable alternative fuel source. For this reason, in recent years biodiesel production has become widespread but this industry currently generates a huge amount of glycerol as a by-product, which has become an environmental problem in its own right. A feasible possibility to solve this problem is the use of waste glycerol as a carbon source for microbial transformation into biofuels such as hydrogen and ethanol. For instance, Escherichia coli is a microorganism that can synthesize these compounds under anaerobic conditions.ResultsIn this work an experimental procedure was established for screening E. coli single mutants to identify strains with enhanced ethanol and/or H2 productions compared to the wild type strain. In an initial screening of 150 single mutants, 12 novel strains (gnd, tdcE, rpiAnanE, tdcB, deoB, sucB, cpsG, frmA, glgC, fumA and gadB) were found to provide enhanced yields for at least one of the target products. The mutations, that improve most significantly the parameters evaluated (gnd and tdcE genes), were combined with other mutations in three engineered E. coli mutant strains in order to further redirect carbon flux towards the desired products.ConclusionsThis methodology can be a useful tool to disclose the metabolic pathways that are more susceptible to manipulation in order to obtain higher molar yields of hydrogen and ethanol using glycerol as main carbon source in multiple E. coli mutants.

Anticentromere antibody specific to human cells directed against the CENP-B autoantigen

We describe the generation of a new antipeptide antibody that binds to the centromeric region of human mitotic chromosomes. This antibody was raised against a synthetic peptide corresponding to the 481-493 amino acid sequence of the human CENP-B autoantigen. Immunofluorescence analysis revealed that this anti-CENP-B serum showed an identical pattern to the human CREST anticentromere autoantibody in both mitotic cells and interphase nuclei. Immunoblotting showed that this antibody reacts with the recombinant human CENP-B autoantigen, indicating that it is directed to the 80-kDa centromere polypeptide. We have used this serum to determine, by indirect immunofluorescence, whether CENP-B is conserved in different mammalian species. Surprisingly, the human antipeptide antibody does not react with the centromeric proteins of cultured mouse, hamster, or Indian muntjac cells. Because the CENP-B gene has been cloned in human and mouse, our results suggest that the CENP-B epitope used as an immunogen in this study is not ubiquitous in mammalian cells, and that we have most probably established a monospecific antibody to the human centromere.

Study of the role played by NfsA, NfsB nitroreductase and NemA flavin reductase from Escherichia coli in the conversion of ethyl 2-(2′-nitrophenoxy)acetate to 4-hydroxy-(2H)-1,4-benzoxazin-3(4H)-one (D-DIBOA), a benzohydroxamic acid with interesting biological properties

Benzohydroxamic acids, such as 4-hydroxy-(2H)-1,4-benzoxazin-3(4H)-one (D-DIBOA), exhibit interesting herbicidal, fungicidal and bactericidal properties. Recently, the chemical synthesis of D-DIBOA has been simplified to only two steps. In a previous paper, we demonstrated that the second step could be replaced by a biotransformation using Escherichia coli to reduce the nitro group of the precursor, ethyl 2-(2′-nitrophenoxy)acetate and obtain D-DIBOA. The NfsA and NfsB nitroreductases and the NemA xenobiotic reductase of E. coli have the capacity to reduce one or two nitro groups from a wide variety of nitroaromatic compounds, which are similar to the precursor. By this reason, we hypothesised that these three enzymes could be involved in this biotransformation. We have analysed the biotransformation yield (BY) of mutant strains in which one, two or three of these genes were knocked out, showing that only in the double nfsA/nfsB and in the triple nfsA/nfsB/nemA mutants, the BY was 0%. These results suggested that NfsA and NfsB are responsible for the biotransformation in the tested conditions. To confirm this, the nfsA and nfsB open reading frames were cloned into the pBAD expression vector and transformed into the nfsA and nfsB single mutants, respectively. In both cases, the biotransformation capacity of the strains was recovered (6.09 ± 0.06% as in the wild-type strain) and incremented considerably when NfsA and NfsB were overexpressed (40.33% ± 9.42% and 59.68% ± 2.0% respectively).

New aspects concerning to the characterization and the relationship with the immune response in vivo of the spiny lobster Panulirus argus nitric oxide synthase.

Nitric oxide (NO) is a short-lived radical generated by nitric oxide synthases (NOS). NO is involved in a variety of functions in invertebrates, including host defense. In a previous study, we isolated and sequenced for the first time the NOS gene from hemocytes of Panulirus argus, demonstrating the inducibility of this enzyme by lipopolysaccharide (LPS) in vitro. In the present work, lobster hemocytes and gills exposed to Escherichia coli O55:B5 LPS showed an increase in both NOS activity and NOS gene expression in vivo. This response was dose and time dependent. The 3D NOS structure was predicted by comparative modeling showing the oxygenase and reductase domains. These domains contain the conserved binding motifs of NOS already found in a variety of organisms. The 3D structure prediction analysis allowed the selection of a fragment of 666bp that was cloned and subsequently expressed in E. coli BL21, in which a recombinant product of around 31KDa was obtained. Hyperimmune serum obtained from immunized rabbits was tested and employed to specifically detect the recombinant polypeptide or the endogenous NOS from lobster hemocytes by western blot and immunofluorescence. This study contributes to enlarge the existing knowledge related to NOS structure and NOS participation in the immune response in lobsters. The evaluation of an antibody capable to recognize NOS from lobsters constitutes a novel and interesting tool for the implementation of further studies on NOS functions in crustaceans.