Programmed cell death and genetic stability in conifer embryogenesis

Somatic embryogenesis, the generation of embryos from somatic cells, is a valuable tool for studying embryology. In addition, somatic embryos can be used for large-scale vegetative propagation, an application of great interest for forestry. A critical event during early embryo differentiation in conifers is the apical basal polarization, which proceeds through the establishment of two embryonic parts: the proliferating embryonal mass and the terminally differentiated suspensor. The development of both parts is strictly coordinated and imbalance causes embryonic defects. The suspensor cells are eliminated by programmed cell death (PCD). In animals, caspase family proteases are the main executioners of PCD. In this work we have used synthetic peptide substrates containing caspase recognition sites and corresponding specific inhibitors to analyse the role of caspase-like activity during early embryo differentiation in Norway spruce (Picea abies L. Karst.). We found that VEIDase is the principal caspase-like activity. This activity is localized specifically in suspensor cells, and its inhibition prevents normal embryo development by blocking the suspensor differentiation. The in vitro VEIDase activity was shown to be highly sensitive to pH, ionic strength, temperature and zinc concentration. In vivo studies with Zinquin, a zinc-specific fluorescent probe, revealed a high accumulation of intracellular free zinc in the embryonal masses and an abrupt decrease in the suspensor. Increased zinc concentration in the culture medium suppresses terminal differentiation and PCD of the suspensor. In accordance, exposure of early embryos to TPEN, a zinc-specific chelator, induces ectopic cell death affecting embryonal masses. This establishes zinc as an important factor affecting cell fate specification during plant embryogenesis. Before somatic embryos can be accepted for clonal propagation it is important to show that the regenerated plants have similar growth to that of seedlings and are genetically uniform. The genetic integrity during zygotic and somatic embryogenesis in Norway spruce and Scots pine (Pinus sylvestris L.) was investigated by comparing the stability of variable nuclear microsatellite loci. The stability varied significantly among families in both species during somatic embryogenesis. Scots pine families showing low genetic stability during establishment of embryogenic cultures had a higher embryogenic potential than those that were genetically more stable. In contrast, embryo development was suppressed in genetically unstable families. The stability of microsatellites was in general higher in zygotic embryos than in somatic embryos. No deviation in growth was observed in somatic embryo plants of Norway spruce carrying mutated microsatellites

The nucleotide and partial amino acid sequences of rat fetuin

Fetuins are among the major plasma proteins, yet their biological role has remained elusive. Here we report the molecular cloning of rat fetuin and the sequence analysis of a full-length clone, RF619 of 1456 bp with an open reading frame of 1056 bp encoding 352 amino acid residues. The coding part of RF619 was identical with the cDNA sequence of the natural inhibitor of the insulin receptor tyrosine kinase from rat (pp63) except for four substitutions and a single base insertion causing divergence of the predicted protein sequences. Partial amino acid sequences of rat plasma fetuin were in agreement with the predictions based on the RF619 cDNA. Purified rat fetuin inhibited the insulin receptor tyrosine kinase in vitro. Therefore, we conclude that RF619 and pp63 cDNA encode the same protein, i.e. authentic rat fetuin which is a functional tyrosine kinase inhibitor

The SARS-coronavirus-host interactome

Coronaviruses (CoVs) are important human and animal pathogens that induce fatal respiratory, gastrointestinal and neurological disease. The outbreak of the severe acute respiratory syndrome (SARS) in 2002/2003 has demonstrated human vulnerability to (Coronavirus) CoV epidemics. Neither vaccines nor therapeutics are available against human and animal CoVs. Knowledge of host cell proteins that take part in pivotal virus-host interactions could define broad-spectrum antiviral targets. In this study, we used a systems biology approach employing a genome-wide yeast-two hybrid interaction screen to identify immunopilins (PPIA, PPIB, PPIH, PPIG, FKBP1A, FKBP1B) as interaction partners of the CoV non-structural protein 1 (Nsp1). These molecules modulate the Calcineurin/NFAT pathway that plays an important role in immune cell activation. Overexpression of NSP1 and infection with live SARS-CoV strongly increased signalling through the Calcineurin/NFAT pathway and enhanced the induction of interleukin 2, compatible with late-stage immunopathogenicity and long-term cytokine dysregulation as observed in severe SARS cases. Conversely, inhibition of cyclophilins by cyclosporine A (CspA) blocked the replication of CoVs of all genera, including SARS-CoV, human CoV-229E and -NL-63, feline CoV, as well as avian infectious bronchitis virus. Non-immunosuppressive derivatives of CspA might serve as broad-range CoV inhibitors applicable against emerging CoVs as well as ubiquitous pathogens of humans and livestock

On a model mechanism for the spatial patterning of teeth primordia in the Alligator

We propose a model mechanism for the initiation and spatial positioning of teeth primordia in the alligator,Alligator mississippiensis. Detailed embryological studies by Westergaard & Ferguson (1986, 1987, 1990) show that jaw growth plays a crucial role in the developmental patterning of the tooth initiation process. Based on biological data we develop a reaction-diffusion mechanism, which crucially includes domain growth. The model can reproduce the spatial pattern development of the first seven teeth primordia in the lower half jaw ofA. mississippiensis. The results for the precise spatio-temporal sequence compare well with detailed developmental experiments

Functional genomic analysis of C. elegans molting.

Although the molting cycle is a hallmark of insects and nematodes, neither the endocrine control of molting via size, stage, and nutritional inputs nor the enzymatic mechanism for synthesis and release of the exoskeleton is well understood. Here, we identify endocrine and enzymatic regulators of molting in C. elegans through a genome-wide RNA-interference screen. Products of the 159 genes discovered include annotated transcription factors, secreted peptides, transmembrane proteins, and extracellular matrix enzymes essential for molting. Fusions between several genes and green fluorescent protein show a pulse of expression before each molt in epithelial cells that synthesize the exoskeleton, indicating that the corresponding proteins are made in the correct time and place to regulate molting. We show further that inactivation of particular genes abrogates expression of the green fluorescent protein reporter genes, revealing regulatory networks that might couple the expression of genes essential for molting to endocrine cues. Many molting genes are conserved in parasitic nematodes responsible for human disease, and thus represent attractive targets for pesticide and pharmaceutical development

A large-scale analysis of mRNAs expressed by primary mesenchyme cells of the sea urchin embryo

The primary mesenchyme cells (PMCs) of the sea urchin embryo have been an important model system for the analysis of cell behavior during gastrulation. To gain an improved understanding of the molecular basis of PMC behavior, a set of 8293 expressed sequenced tags (ESTs) was derived from an enriched population of mid-gastrula stage PMCs. These ESTs represented approximately 1200 distinct proteins, or about 15% of the mRNAs expressed by the gastrula stage embryo. 655 proteins were similar (P<10-7 by BLAST comparisons) to other proteins in GenBank, for which some information is available concerning expression and/or function. Another 116 were similar to ESTs identified in other organisms, but not further characterized. We conservatively estimate that sequences encoding at least 435 additional proteins were included in the pool of ESTs that did not yield matches by BLAST analysis. The collection of newly identified proteins includes many candidate regulators of primary mesenchyme morphogenesis, including PMC-specific extracellular matrix proteins, cell surface proteins, spicule matrix proteins and transcription factors. This work provides a basis for linking specific molecular changes to specific cell behaviors during gastrulation. Our analysis has also led to the cloning of several key components of signaling pathways that play crucial roles in early sea urchin development

Identification of novel amplification gene targets in mouse and human breast cancer at a syntenic cluster mapping to mouse identification of novel amplification gene targets in mouse and human breast cancer at a syntenic cluster mapping to mouse ch8a1 and human ch13q34

Serial analysis of gene expression from aggressive mammary tumors derived from transplantable p53 null mouse mammary outgrowth lines revealed significant up-regulation of Tfdp1 (transcription factor Dp1), Lamp1 (lysosomal membrane glycoprotein 1) and Gas6 (growth arrest specific 6) transcripts. All of these genes belong to the same linkage cluster, mapping to mouse chromosome band 8A1. BAC-array comparative genomic hybridization and fluorescence in situ hybridization analyses revealed genomic amplification at mouse region ch8A1.1. The minimal region of amplification contained genes Cul4a, Lamp1, Tfdp1, and Gas6, highly overexpressed in the p53 null mammary outgrowth lines at preneoplastic stages, and in all its derived tumors. The same amplification was also observed in spontaneous p53 null mammary tumors. Interestingly, this region is homologous to human chromosome 13q34, and some of the same genes were previously observed amplified in human carcinomas. Thus, we further investigated the occurrence and frequency of gene amplification affecting genes mapping to ch13q34 in human breast cancer. TFDP1 showed the highest frequency of amplification affecting 31% of 74 breast carcinomas analyzed. Statistically significant positive correlation was observed for the amplification of CUL4A, LAMP1, TFDP1, and GAS6 genes (P < 0.001). Meta-analysis of publicly available gene expression data sets showed a strong association between the high expression of TFDP1 and decreased overall survival (P = 0.00004), relapse-free survival (P = 0.0119), and metastasis-free interval (P = 0.0064). In conclusion, our findings suggest that CUL4A, LAMP1, TFDP1, and GAS6 are targets for overexpression and amplification in breast cancers. Therefore, overexpression of these genes and, in particular, TFDP1 might be of relevance in the development and/or progression in a significant subset of human breastFil: Abba, Martín Carlos. University of Texas; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Fabris, Victoria Teresa. University of Texas; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Hu, Yuhui. University of Texas; Estados UnidosFil: Kittrell, Frances S.. Baylor College of Medicine; Estados Unidos. University of Texas; Estados UnidosFil: Cai, Wei Wen. University of Texas; Estados Unidos. Baylor College of Medicine; Estados UnidosFil: Donehower, Lawrence A.. University of Texas; Estados UnidosFil: Sahin, Aysegui. University of Texas; Estados UnidosFil: Medina, Daniel. University of Texas; Estados Unidos. Baylor College of Medicine; Estados UnidosFil: Aldaz, Claudio Marcelo. University of Texas; Estados Unido