Exploring the Origin and Physiological Significance of DNA Double Strand Breaks in the Developing Neuroretina.

Genetic mosaicism is an intriguing physiological feature of the mammalian brain that generates altered genetic information and provides cellular, and prospectively functional, diversity in a manner similar to that of the immune system. However, both its origin and its physiological significance remain poorly characterized. Most, if not all, cases of somatic mosaicism require prior generation and repair of DNA double strand breaks (DSBs). The relationship between DSB generation, neurogenesis, and early neuronal cell death revealed by our studies in the developing retina provides new perspectives on the different mechanisms that contribute to DNA rearrangements in the developing brain. Here, we speculate on the physiological significance of these findings.

Intracellular Mechanical Drugs Induce Cell-Cycle Altering and Cell Death.

Current advances in materials science have demonstrated that extracellular mechanical cues can define cell function and cell fate. However, a fundamental understanding of the manner in which intracellular mechanical cues affect cell mechanics remains elusive. How intracellular mechanical hindrance, reinforcement, and supports interfere with the cell cycle and promote cell death is described here. Reproducible devices with highly controlled size, shape, and with a broad range of stiffness are internalized in HeLa cells. Once inside, they induce characteristic cell-cycle deviations and promote cell death. Device shape and stiffness are the dominant determinants of mechanical impairment. Device structural support to the cell membrane and centering during mitosis maximize their effects, preventing spindle centering, and correct chromosome alignment. Nanodevices reveal that the spindle generates forces larger than 114 nN which overcomes intracellular confinement by relocating the device to a less damaging position. By using intracellular mechanical drugs, this work provides a foundation to defining the role of intracellular constraints on cell function and fate, with relevance to fundamental cell mechanics and nanomedicine.

Integrating magnetic capabilities to intracellular chips for cell trapping.

Current microtechnologies have shown plenty of room inside a living cell for silicon chips. Microchips as barcodes, biochemical sensors, mechanical sensors and even electrical devices have been internalized into living cells without interfering their cell viability. However, these technologies lack from the ability to trap and preconcentrate cells in a specific region, which are prerequisites for cell separation, purification and posterior studies with enhanced sensitivity. Magnetic manipulation of microobjects, which allows a non-contacting method, has become an attractive and promising technique at small scales. Here, we show intracellular Ni-based chips with magnetic capabilities to allow cell enrichment. As a proof of concept of the potential to integrate multiple functionalities on a single device of this technique, we combine coding and magnetic manipulation capabilities in a single device. Devices were found to be internalized by HeLa cells without interfering in their viability. We demonstrated the tagging of a subpopulation of cells and their subsequent magnetic trapping with internalized barcodes subjected to a force up to 2.57 pN (for magnet-cells distance of 4.9 mm). The work opens the venue for future intracellular chips that integrate multiple functionalities with the magnetic manipulation of cells.

Reversals in complex traits uncovered as reticulation events: Lessons from the evolution of parity-mode, chromosome morphology, and maternal resource transfer.

Complex traits include, among many others, the evolution of eyes, wings, body forms, reproductive modes, human intelligence, social behavior, diseases, and chromosome morphology. Dollo's law states that the evolution of complex traits is irreversible. However, potential exceptions have been proposed. Here, we investigated whether reticulation, a simple and elegant means by which complex characters may be reacquired, could account for suggested reversals in the evolution of complex characters using two datasets with sufficient genetic coverage and a total of five potential reversals. Our analyses uncovered a potential reversal in the evolution of parity mode and a potential reversal in the evolution of placentotrophy of fish (Cyprinodontiformes) as reticulation events. Moreover, in a reptile that exhibits a potential reversal from viviparity to oviparity (Zootoca vivipara), reticulation provided the most parsimonious explanation for sex chromosome evolution. Therefore, three of the five studied potential reversals were unraveled as reticulation events. This constitutes the first evidence that accounting for reticulation can fundamentally influence the interpretation of the evolution of complex traits, that testing for reticulation is crucial for obtaining robust phylogenies, and that complex ancestral characters may be reacquired through hybridization with a lineage that still exhibits the trait. Hybridization, rather than reappearance of ancestral traits by means of small evolutionary steps, may thus account for suggested exceptions to Dollo's law. Consequently, ruling out reticulation is required to claim the evolutionary reversal of complex characters and potential exceptions to Dollo's rule.

RAG-2 deficiency results in fewer phosphorylated histone H2AX foci, but increased retinal ganglion cell death and altered axonal growth.

DNA double-strand breaks (DSBs), selectively visualized as γ-H2AX+ foci, occur during the development of the central nervous system, including the retina, although their origin and biological significance are poorly understood. Mutant mice with DSB repair mechanism defects exhibit increased numbers of γ-H2AX+ foci, increased cell death during neural development, and alterations in axonogenesis in the embryonic retina. The aim of this study was to identify putative sources of DSBs. One of the identified DSBs sources is LINE-1 retrotransposition. While we did not detect changes in LINE-1 DNA content during the early period of cell death associated with retinal neurogenesis, retinal development was altered in mice lacking RAG-2, a component of the RAG-1,2-complex responsible for initiating somatic recombination in lymphocytes. Although γ-H2AX+ foci were less abundant in the rag2-/- mouse retina, retinal ganglion cell death was increased and axonal growth and navigation were impaired in the RAG-2 deficient mice, a phenotype shared with mutant mice with defective DNA repair mechanisms. These findings demonstrate that RAG-2 is necessary for proper retinal development, and suggest that both DSB generation and repair are genuine processes intrinsic to neural development.

Stalk cell differentiation without polyketides in the cellular slime mold.

Polyketides induce prestalk cell differentiation in Dictyostelium. In the double-knockout mutant of the SteelyA and B polyketide synthases, most of the pstA cells-the major part of the prestalk cells-are lost, and we show by whole mount in situ hybridization that expression of prestalk genes is also reduced. Treatment of the double-knockout mutant with the PKS inhibitor cerulenin gave a further reduction, but some pstA cells still remained in the tip region, suggesting the existence of a polyketide-independent subtype of pstA cells. The double-knockout mutant and cerulenin-treated parental Ax2 cells form fruiting bodies with fragile, single-cell layered stalks after cerulenin treatment. Our results indicate that most pstA cells are induced by polyketides, but the pstA cells at the very tip of the slug are induced in some other way. In addition, a fruiting body with a single-cell layered, vacuolated stalk can form without polyketides.

Alternative Eukaryotic Expression Systems for the Production of Proteins and Protein Complexes.

Besides the most established expression hosts, several eukaryotic microorganisms and filamentous fungi have also been successfully used as platforms for the production of foreign proteins. Filamentous fungi and Dictyostelium discoideum are two prominent examples. Filamentous fungi, typically Aspergillus and Trichoderma, are usually employed for the industrial production of enzymes and secondary metabolites for food processing, pharmaceutical drugs production, and textile and paper applications, with multiple products already accepted for their commercialization. The low cost of culture medium components, high secretion capability directly to the extracellular medium, and the intrinsic ability to produce post-translational modifications similar to the mammalian type, have promoted this group as successful hosts for the expression of proteins, including examples from phylogenetically distant groups: humans proteins such as IL-2, IL-6 or epithelial growth factor; α-galactosidase from plants; or endoglucanase from Cellulomonas fimi, among others. D. discoideum is a social amoeba that can be used as an expression platform for a variety of proteins, which has been extensively illustrated for cytoskeletal proteins. New vectors for heterologous expression in D. discoideum have been recently developed that might increase the usefulness of this system and expand the range of protein classes that can be tackled. Continuous developments are ongoing to improve strains, promoters, production and downstream processes for filamentous fungi, D. discoideum, and other alternative eukaryotic hosts. Either for the overexpression of individual genes, or in the coexpression of multiples genes, this chapter illustrates the enormous possibilities offered by these groups of eukaryotic organisms.

Increased neuronal death and disturbed axonal growth in the Polµ-deficient mouse embryonic retina.

Programmed cell death occurs naturally at different stages of neural development, including neurogenesis. The functional role of this early phase of neural cell death, which affects recently differentiated neurons among other cell types, remains undefined. Some mouse models defective in DNA double-strand break (DSB) repair present massive cell death during neural development, occasionally provoking embryonic lethality, while other organs and tissues remain unaffected. This suggests that DSBs occur frequently and selectively in the developing nervous system. We analyzed the embryonic retina of a mouse model deficient in the error-prone DNA polymerase µ (Polµ), a key component of the non-homologous end-joining (NHEJ) repair system. DNA DSBs were increased in the mutant mouse at embryonic day 13.5 (E13.5), as well as the incidence of cell death that affected young neurons, including retinal ganglion cells (RGCs). Polµ(-/-) mice also showed disturbed RGC axonal growth and navigation, and altered distribution of the axonal guidance molecules L1-CAM and Bravo (also known as Nr-CAM). These findings demonstrate that Polµ is necessary for proper retinal development, and support that the generation of DSBs and their repair via the NHEJ pathway are genuine processes involved in neural development.

Suspended Planar-Array Chips for Molecular Multiplexing at the Microscale.

A novel suspended planar-array chips technology is described, which effectively allows molecular multiplexing using a single suspended chip to analyze extraordinarily small volumes. The suspended chips are fabricated by combining silicon-based technology and polymer-pen lithography, obtaining increased molecular pattern flexibility, and improving miniaturization and parallel production. The chip miniaturization is so dramatic that it permits the intracellular analysis of living cells.

Silicon chips detect intracellular pressure changes in living cells.

The ability to measure pressure changes inside different components of a living cell is important, because it offers an alternative way to study fundamental processes that involve cell deformation. Most current techniques such as pipette aspiration, optical interferometry or external pressure probes use either indirect measurement methods or approaches that can damage the cell membrane. Here we show that a silicon chip small enough to be internalized into a living cell can be used to detect pressure changes inside the cell. The chip, which consists of two membranes separated by a vacuum gap to form a Fabry-Pérot resonator, detects pressure changes that can be quantified from the intensity of the reflected light. Using this chip, we show that extracellular hydrostatic pressure is transmitted into HeLa cells and that these cells can endure hypo-osmotic stress without significantly increasing their intracellular hydrostatic pressure.

The NMRA/NMRAL1 homologue PadA modulates the expression of extracellular cAMP relay genes during aggregation in Dictyostelium discoideum.

NMRA-like proteins belong to a class of conserved transcriptional regulators that function as direct sensors of the metabolic state of the cell and link basic metabolism to changes in gene expression. PadA was the first NMRA-like protein described in Dictyostelium discoideum and was shown to be necessary for prestalk cell differentiation and correct development. We describe and characterize padA(-) mutant phenotype during the onset of development, which results in the formation of abnormally small territories and impairment of cAMP responses. Transcriptional analysis shows that cAMP-induced gene expression is downregulated in padA(-), particularly the genes that establish the extracellular cAMP relay. The mutant phenotype can be rescued with the constitutive expression of one of these genes, carA, encoding the cAMP receptor. Transcriptional analysis of padA(-)/A15::carA showed that carA maximum mRNA levels were not reached during aggregation. Our data support a regulatory role for PadA on the regulation of extracellular cAMP relay genes during aggregation and suggest that PadA is required to achieve carA full induction.

Mef2A, a homologue of animal Mef2 transcription factors, regulates cell differentiation in Dictyostelium discoideum.

BACKGROUND: Transcription factors from the MADS-box family play a relevant role in cell differentiation and development and include the animal SRF (serum response factor) and MEF2 (myocyte enhancer factor 2) proteins. The social amoeba Dictyostelium discoideum contains four genes coding for MADS-box transcription factors, two of these genes code for proteins that are more similar to SRF, and the other two code for proteins that are more similar to MEF2 animal factors. RESULTS: The biological function of one of the two genes that codes for MEF2-related proteins, a gene known as mef2A, is described in this article. This gene is expressed under the transcriptional control of two alternative promoters in growing cells, and its expression is induced during development in prespore cells. Mutant strains where the mef2A gene has been partially deleted were generated to study its biological function. The mutant strains showed reduced growth when feeding on bacteria and were able to develop and form fruiting bodies, but spore production was significantly reduced. A study of developmental markers showed that prespore cells differentiation was impaired in the mutant strains. When mutant and wild-type cells were set to develop in chimeras, mutant spores were underrepresented in the fruiting bodies. The mutant cells were also unable to form spores in vitro. In addition, mutant cells also showed a poor contribution to the formation of the tip-organizer and the upper region of slugs and culminant structures. In agreement with these observations, a comparison of the genes transcribed by mutant and wild-type strains during development indicated that prestalk gene expression was enhanced, while prespore gene expression decreased in the mef2A- strain. CONCLUSIONS: Our data shows that mef2A plays a role in cell differentiation in D. discoideum and modulates the expression of prespore and prestalk genes.

Role of bacterial surface structures on the interaction of Klebsiella pneumoniae with phagocytes.

Phagocytosis is a key process of the immune system. The human pathogen Klebsiella pneumoniae is a well known example of a pathogen highly resistant to phagocytosis. A wealth of evidence demonstrates that the capsule polysaccharide (CPS) plays a crucial role in resistance to phagocytosis. The amoeba Dictyostelium discoideum shares with mammalian macrophages the ability to phagocytose and kill bacteria. The fact that K. pneumoniae is ubiquitous in nature and, therefore, should avoid predation by amoebae, poses the question whether K. pneumoniae employs similar means to counteract amoebae and mammalian phagocytes. Here we developed an assay to evaluate K. pneumoniae-D. discoideum interaction. The richness of the growth medium affected the threshold at which the cps mutant was permissive for Dictyostelium and only at lower nutrient concentrations the cps mutant was susceptible to predation by amoebae. Given the critical role of bacterial surface elements on host-pathogen interactions, we explored the possible contribution of the lipopolysaccharide (LPS) and outer membrane proteins (OMPs) to combat phagoyctosis by D. discoideum. We uncover that, in addition to the CPS, the LPS O-polysaccharide and the first core sugar participate in Klebsiella resistance to predation by D. discoideum. K. pneumoniae LPS lipid A decorations are also necessary to avoid predation by amoebae although PagP-dependent palmitoylation plays a more important role than the lipid A modification with aminoarabinose. Mutants lacking OMPs OmpA or OmpK36 were also permissive for D. discoideium growth. Except the LPS O-polysaccharide mutants, all mutants were more susceptible to phagocytosis by mouse alveolar macrophages. Finally, we found a correlation between virulence, using the pneumonia mouse model, and resistance to phagocytosis. Altogether, this work reveals novel K. pneumoniae determinants involved in resistance to phagocytosis and supports the notion that Dictyostelium amoebae might be useful as host model to measure K. pneumoniae virulence and not only phagocytosis.

Integrative analyses of speciation and divergence in Psammodromus hispanicus (Squamata: Lacertidae).

BACKGROUND: Genetic, phenotypic and ecological divergence within a lineage is the result of past and ongoing evolutionary processes, which lead ultimately to diversification and speciation. Integrative analyses allow linking diversification to geological, climatic, and ecological events, and thus disentangling the relative importance of different evolutionary drivers in generating and maintaining current species richness. RESULTS: Here, we use phylogenetic, phenotypic, geographic, and environmental data to investigate diversification in the Spanish sand racer (Psammodromus hispanicus). Phylogenetic, molecular clock dating, and phenotypic analyses show that P. hispanicus consists of three lineages. One lineage from Western Spain diverged 8.3 (2.9-14.7) Mya from the ancestor of Psammodromus hispanicus edwardsianus and P. hispanicus hispanicus Central lineage. The latter diverged 4.8 (1.5-8.7) Mya. Molecular clock dating, together with population genetic analyses, indicate that the three lineages experienced northward range expansions from southern Iberian refugia during Pleistocene glacial periods. Ecological niche modelling shows that suitable habitat of the Western lineage and P. h. edwardsianus overlap over vast areas, but that a barrier may hinder dispersal and genetic mixing of populations of both lineages. P. h. hispanicus Central lineage inhabits an ecological niche that overlaps marginally with the other two lineages. CONCLUSIONS: Our results provide evidence for divergence in allopatry and niche conservatism between the Western lineage and the ancestor of P. h. edwardsianus and P. h. hispanicus Central lineage, whereas they suggest that niche divergence is involved in the origin of the latter two lineages. Both processes were temporally separated and may be responsible for the here documented genetic and phenotypic diversity of P. hispanicus. The temporal pattern is in line with those proposed for other animal lineages. It suggests that geographic isolation and vicariance played an important role in the early diversification of the group, and that lineage diversification was further amplified through ecological divergence.

The Dictyostelium discoideum acaA gene is transcribed from alternative promoters during aggregation and multicellular development.

BACKGROUND: Extracellular cAMP is a key extracellular signaling molecule that regulates aggregation, cell differentiation and morphogenesis during multi-cellular development of the social amoeba Dictyostelium discoideum. This molecule is produced by three different adenylyl cyclases, encoded by the genes acaA, acrA and acgA, expressed at different stages of development and in different structures. METHODOLOGY/PRINCIPAL FINDINGS: This article describes the characterization of the promoter region of the acaA gene, showing that it is transcribed from three different alternative promoters. The distal promoter, promoter 1, is active during the aggregation process while the more proximal promoters are active in tip-organiser and posterior regions of the structures. A DNA fragment containing the three promoters drove expression to these same regions and similar results were obtained by in situ hybridization. Analyses of mRNA expression by quantitative RT-PCR with specific primers for each of the three transcripts also demonstrated their different temporal patterns of expression. CONCLUSIONS/SIGNIFICANCE: The existence of an aggregation-specific promoter can be associated with the use of cAMP as chemo-attractant molecule, which is specific for some Dictyostelium species. Expression at late developmental stages indicates that adenylyl cyclase A might play a more important role in post-aggregative development than previously considered.

Eisosome organization in the filamentous ascomycete Aspergillus nidulans.

Eisosomes are subcortical organelles implicated in endocytosis and have hitherto been described only in Saccharomyces cerevisiae. They comprise two homologue proteins, Pil1 and Lsp1, which colocalize with the transmembrane protein Sur7. These proteins are universally conserved in the ascomycetes. We identify in Aspergillus nidulans (and in all members of the subphylum Pezizomycotina) two homologues of Pil1/Lsp1, PilA and PilB, originating from a duplication independent from that extant in the subphylum Saccharomycotina. In the aspergilli there are several Sur7-like proteins in each species, including one strict Sur7 orthologue (SurG in A. nidulans). In A. nidulans conidiospores, but not in hyphae, the three proteins colocalize at the cell cortex and form tightly packed punctate structures that appear different from the clearly distinct eisosome patches observed in S. cerevisiae. These structures are assembled late during the maturation of conidia. In mycelia, punctate structures are present, but they are composed only of PilA, while PilB is diffused in the cytoplasm and SurG is located in vacuoles and endosomes. Deletion of each of the genes does not lead to any obvious growth phenotype, except for moderate resistance to itraconazole. We could not find any obvious association between mycelial (PilA) eisosome-like structures and endocytosis. PilA and SurG are necessary for conidial eisosome organization in ways that differ from those for their S. cerevisiae homologues. These data illustrate that conservation of eisosomal proteins within the ascomycetes is accompanied by a striking functional divergence.

Idiopathic pulmonary fibrosis and emphysema: decreased survival associated with severe pulmonary arterial hypertension.

BACKGROUND: It has been suggested that the presence of emphysema modifies the outcome of patients with idiopathic pulmonary fibrosis (IPF). In this article we compare clinical features, smoking history, pulmonary function, estimated systolic pulmonary artery pressure (eSPAP), and mortality in IPF with emphysema vs IPF without emphysematous changes. METHODS: A cohort of 110 IPF patients was evaluated. Clinical data were collected from clinical charts. High-resolution CT (HRCT) scans were examined by an expert blinded to clinical data, and patients were classified into the following two groups: patients with IPF with emphysema; and patients with IPF without emphysema. The Kaplan-Meier method, log-rank test, and Cox regression model were used for statistical analyses. RESULTS: The prevalence of emphysema in the IPF cohort was 28% (31 of 110 patients). IPF with emphysema was significantly associated with male gender (odds ratio [OR], 18; 95% confidence interval [CI], 2.7 to 773.7; p = 0.0003), and smoking (OR, 3.8; 95% CI, 1.36 to 11.6; p = 0.004). Patients with IPF and emphysema had a higher mean (+/- SD) decrease in oxygen saturation during rest and exercise (16.3 +/- 6.7% vs 13.5 +/- 4.6%, respectively; p = 0.04), a higher mean fibrosis HRCT scan score (1.75 +/- 0.36 vs 1.55 +/- 0.38, respectively; p = 0.015), a higher eSPAP (82 +/- 20 vs 57 +/- 15 mm Hg, respectively; p < 0.0001), and lower median survival time (25 vs 34 months, respectively; p = 0.01) than patients with IPF without emphysema. The Cox regression model showed that the two most important variables associated with mortality were FVC < 50% predicted (hazard ratio [HR], 2.6; 95% CI, 1.19 to 5.68; p = 0.016) and eSPAP >or= 75 mm Hg (HR, 2.25; 95% CI, 1.12 to 4.54; p = 0.022). CONCLUSIONS: IPF patients with emphysema exhibited higher mortality compared with those with IPF without emphysema. This dire prognosis seems to be at least partially associated with the development of severe pulmonary arterial hypertension.

Utilidad de los marcadores tumorales en líquidos de punción pleural / Usefulness of tumor markers in pleural fluids

El diagnóstico de la neoplasia pleural se basa en la demostración de células neoplásicas en fluido pleural (FP) o en biopsias de pleura. Sin embargo, aún en casos de malignidad hay un elevado porcentaje de informes falsos negativos (30-60%). Los FP neoplásicos presentan valores detectables de marcadores tumorales (MT) producidos por las células neoplásicas de la pleura. El objetivo de este trabajo ha sido evaluar la utilidad diagnóstica de algunos marcadores tumorales, de uso corriente en el laboratorio, en los fluidos de punción pleural. En 20 de ellos se analizaron: Antígeno cárcinoembrionario (CEA), fragmentos de la citoqueratina 19 (CYFRA 21-1) y Antígeno CA 125. Se efectuó el estudio fisicoquímico, recuento celular y examen citológico (Papanicolaou). En algunos casos se realizó diagnóstico anátomo-patológico. Los MT se dosaron por inmunoensayo de electroquimioluminiscencia. La comparación se efectuó por Kruskal-Wallis. Las muestras fueron clasificadas en 4 grupos y se determinó en cada uno mediana y rango para CEA (ng/mL), CYFRA (ng/mL) y CA 125 (UI/mL), respectivamente: 1) Citología positiva con diagnóstico previo de cáncer de pulmón (n: 5) 112 (2,3-1.610), 134,4 (45,8-600), 1.048 (498-2999); 2) Citología positiva con diagnóstico previo de cáncer de otro origen (n: 4) 15,28 (1,1-93,80), 108,1 (7,42-497,2), 1.827 (1.103-14.130); 3) Citología negativa con diagnóstico incierto (n: 4) 1,89 (0,91-2,96), 17,1 (1,5-29,6), 578,2 (27,8-12); 4) Citología no concluyente con diagnóstico incierto (n: 7) 31,8 (1,28-370,2), 96,3 (23,8-860), 585 (94,4-4.584). Se observó diferencia significativa entre los grupos. La combinación de citología y MT aumentó el diagnóstico de neoplasia pleural en 25%. Los resultados preliminares permiten concluir que un panel de marcadores tumorales en FP, sumado a los estudios tradicionales, representa una ayuda diagnóstica.

The diagnosis of pleural cancer is supported by the demonstration of neoplastic cells in pleural fluid or in pleural biopsies. However, even in malignancy there are a great number of false negatives results (30-60%). Tumor fluids. To establish the value of different tumor markers, frequently used in clinical laboratories, in the diagnosis of pleural fluids. 20 pleural fluids were processed for physical and chemical study, cellular counting, morphological examination (Papanicolaou stain) and electrochemiluminescense immunoassay for carcinoembryonic antigen (CEA), CYFRA 21-1 and CA 125. The results were analysed by Kruskal-Wallis. In some cases, biopsies were performed. The samples were classified in four groups, and the median and rank were calculated in each case (CEA, CYFRA and CA 125). 1) Positive cytology with previous diagnosis of lung cancer (n: 5) 112 (2.3-1610), 134.4 (45.8-600), 1048 (498-2999) 2) Positive cytology with previous diagnosis of not-lung cancer(n: 4) 15.28 (1.1-93.80), 108.1 (7.42- 497.2),1827 (1103-14130), 3) Negative cytology with uncertain diagnosis (n: 4)1.89 (0.91- 2.96), 17.1 (1.5-29.6), 578.2 (27.8-12, 4) Inconclusive cytology with uncertain diagnosis (n: 7) 31.8 (1.28-370.2), 96.3 (23.8-860), 585 (94.4 -4584). There were stastistic differences among the four groups. Joining the cytology to the assays for tumor markers raised sensitivity by 25%. The assay for tumor markers can be a complementary tool in the diagnosis of effusions.

A new protein carrying an NmrA-like domain is required for cell differentiation and development in Dictyostelium discoideum.

We have isolated a Dictyostelium mutant unable to induce expression of the prestalk-specific marker ecmB in monolayer assays. The disrupted gene, padA, leads to a range of phenotypic defects in growth and development. We show that padA is essential for growth, and we have generated a thermosensitive mutant allele, padA(-). At the permissive temperature, mutant cells grow poorly; they remain longer at the slug stage during development and are defective in terminal differentiation. At the restrictive temperature, growth is completely blocked, while development is permanently arrested prior to culmination. padA(-) slugs are deficient in prestalk A cell differentiation and present an abnormal ecmB expression pattern. Sequence comparisons and predicted three-dimensional structure analyses show that PadA carries an NmrA-like domain. NmrA is a negative transcriptional regulator involved in nitrogen metabolite repression in Aspergillus nidulans. PadA predicted structure shows a NAD(P)(+)-binding domain, which we demonstrate that is essential for function. We show that padA(-) development is more sensitive to ammonia than wild-type cells and two ammonium transporters, amtA and amtC, appear derepressed during padA(-) development. Our data suggest that PadA belongs to a new family of NAD(P)(+)-binding proteins that link metabolic changes to gene expression and is required for growth and normal development.