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To provide accurate biological hypotheses and elucidate global properties of cellular networks, systematic identification of protein-protein interactions must meet high quality standards.We present an expanded C. elegans protein-protein interaction network, or 'interactome' map, derived from testing a matrix of approximately 10,000 x approximately 10,000 proteins using a highly specific, high-throughput yeast two-hybrid system. Through a new empirical quality control framework, we show that the resulting data set (Worm Interactome 2007, or WI-2007) was similar in quality to low-throughput data curated from the literature. We filtered previous interaction data sets and integrated them with WI-2007 to generate a high-confidence consolidated map (Worm Interactome version 8, or WI8). This work allowed us to estimate the size of the worm interactome at approximately 116,000 interactions. Comparison with other types of functional genomic data shows the complementarity of distinct experimental approaches in predicting different functional relationships between genes or proteins

Many quantitative trait loci (QTLs) for quality traits have been located on the tomato genetic map, but introgression of favourable wild alleles into large fruited species is hampered by co-localizations of QTLs with antagonist effects. The aim of this study was to assess the growth processes controlled by the main QTLs for fruit size and composition. Four nearly isogenic lines (NILs) derived from an intraspecific cross between a tasty cherry tomato (Cervil) and a normal-tasting large fruit tomato (Levovil) were studied. The lines carried one (L2, L4, and L9) or five (Lx) introgressions from Cervil on chromosomes 1, 2, 4, and 9. QTLs for fruit size could be mainly associated with cell division processes in L2 and L9, whereas cell expansion was rather homogeneous among the genotypes, except Cervil for which the low expansion rate was attributed to low cell plasticity. The link between endoreduplication and fruit size remained unclear, as cell or fruit sizes were positively correlated with the cell DNA content, but not with the endoreduplication factor. QTLs for fruit composition reflected differences in water accumulation rather than in sugar accumulation, except in L9 for which the up-regulation of sucrose unloading and hexose transport and/or starch synthesis was suggested. This may explain the increased amount of carbon allocated to cell structures in L9, which could be related to a QTL for fruit texture. In Lx, these effects were attenuated, except on fruit size and cell division. Finally, the region on top of chromosome 9 may control size and composition attributes in tomato, by a combination of QTL effects on cell division, cell wall synthesis, and carbon import and metabolism.

Throughout tomato domestication, a large increase in fruit size was associated with a loss of dry matter and sugar contents. This study aims to dissect the contributions of genetic variation and the physiological processes underlying the relationships between fruit growth and the accumulation of dry matter and sugars. Fruit quality traits and physiological parameters were measured on 20 introgression lines derived from the introgression of Solanum chmielewskii into S. lycopersicum, under high (HL, unpruned trusses) and low (LL, trusses pruned to one fruit) fruit load conditions. Inter- and intra-genotypic correlations among traits were estimated and quantitative trait loci (QTL) for size, composition, and physiological traits were mapped. LL increased almost all traits, but the response of sugar content was genotype-dependent, involving either dilution effects or differences in carbon allocation to sugars. Genotype x fruit load interactions were significant for most traits and only 30% of the QTL were stable under both fruit loads. Many QTL for fresh weight and cell or seed numbers co-localized. Eleven clusters of QTL for fresh weight and dry matter or sugar content were detected, eight with opposite allele effects and three with negative effects. Two genotypic antagonistic relationships, between fresh weight and dry matter content and between cell number and cell size, were significant only under HL; the second could be interpreted as a competition for carbohydrates among cells. The role of cuticular conductance, fruit transpiration or cracking in the relationship between fruit fresh weight and composition was also emphasized at the genetic and physiological levels.

The airways are thought to dilate during exercise in humans but the time course and mechanisms of the response are not fully described. The aim of the study was to document changes in airway calibre during electrically induced muscular contractions (MC) in spontaneously breathing rabbits. Experiments were also performed after induced bronchoconstriction to assess the effect of change in breathing pattern on airway calibre during MC. Respiratory resistance (R(RS)) was measured in 12 rabbits using the forced oscillation technique at 20 Hz before, during and after 30s MC in control conditions and after methacholine induced bronchoconstriction (Mch). MC was associated with significant decrease in R(RS) both at control and Mch. The MC induced increase in V(E) occurred with significant albeit small change in mean tidal volume (V(T)) at Mch but not control. An augmented breath (AB) occurred in 29/35 MCs and was usually associated with an abrupt drop in R(RS). The decrease in R(RS) induced by AB was significantly larger at Mch compared with control. Passively inflating the lung after MC induced significantly larger decrease in R(RS) than AB during MC. The data indicate bronchodilation by MC in spontaneously breathing rabbits. The mechanisms appear to include AB dependent airway wall stretching as well as removal of cholinergic input to the airway smooth muscle.

Many cancer-associated genes remain to be identified to clarify the underlying molecular mechanisms of cancer susceptibility and progression. Better understanding is also required of how mutations in cancer genes affect their products in the context of complex cellular networks. Here we have used a network modeling strategy to identify genes potentially associated with higher risk of breast cancer. Starting with four known genes encoding tumor suppressors of breast cancer, we combined gene expression profiling with functional genomic and proteomic (or 'omic') data from various species to generate a network containing 118 genes linked by 866 potential functional associations. This network shows higher connectivity than expected by chance, suggesting that its components function in biologically related pathways. One of the components of the network is HMMR, encoding a centrosome subunit, for which we demonstrate previously unknown functional associations with the breast cancer-associated gene BRCA1. Two case-control studies of incident breast cancer indicate that the HMMR locus is associated with higher risk of breast cancer in humans. Our network modeling strategy should be useful for the discovery of additional cancer-associated genes.

Differential regulation of gene expression is essential for cell fate specification in metazoans. Characterizing the transcriptional activity of gene promoters, in time and in space, is therefore a critical step toward understanding complex biological systems. Here we present an in vivo spatiotemporal analysis for approximately 900 predicted C. elegans promoters (approximately 5% of the predicted protein-coding genes), each driving the expression of green fluorescent protein (GFP). Using a flow-cytometer adapted for nematode profiling, we generated 'chronograms', two-dimensional representations of fluorescence intensity along the body axis and throughout development from early larvae to adults. Automated comparison and clustering of the obtained in vivo expression patterns show that genes coexpressed in space and time tend to belong to common functional categories. Moreover, integration of this data set with C. elegans protein-protein interactome data sets enables prediction of anatomical and temporal interaction territories between protein partners.

Tomato (Solanum lycopersicum) is a model plant for studying fleshy fruit development. Several genetic and molecular approaches have been developed to increase our knowledge about the physiological basis of fruit growth, but very few data are yet available at the proteomic level. The main stages of fruit development were first determined through the dynamics of fruit diameter and pericarp cell number. Then, total proteins were extracted from pericarp tissue at six relevant developmental stages and separated by two-dimensional gel electrophoresis. Protein patterns were markedly different between stages. Proteins showing major variations were monitored. We identified 90 of 1,791 well-resolved spots either by matrix-assisted laser-desorption ionization time-of-flight peptide mass fingerprinting or liquid chromatography-mass spectrometry sequencing and expressed sequence tag database searching. Clustered correlation analysis results pointed out groups of proteins with similar expression profiles during fruit development. In young fruit, spots linked to amino acid metabolism or protein synthesis were mainly expressed during the cell division stage and down-regulated later. Some spots linked to cell division processes could be identified. During the cell expansion phase, spots linked to photosynthesis and proteins linked to cell wall formation transiently increased. In contrast, the major part of the spots related to C compounds and carbohydrate metabolism or oxidative processes were up-regulated during fruit development, showing an increase in spot intensity during development and maximal abundance in mature fruit. This was also the case for spots linked to stress responses and fruit senescence. We discuss protein variations, taking into account their potential role during fruit growth and comparing our results with already known variations at mRNA and metabolite-profiling levels.

Endoreduplication is a phenomenon, widespread among plants, which consists of an incomplete cell cycle without mitosis and leads to the increase of the nuclear DNA content. In this work, a model was developed describing cell proliferation and DNA endoreduplication over the whole fruit development, from the pre-anthesis period until maturation. In each mitotic cycle of duration tau, the proportion of cells proceeding through division depends on a constant parameter rho and on the progressive decline of the proliferating capacity . The non-dividing cells may either stop the reduplication fully, or switch to repeated syntheses of DNA without cell division, resulting in cell endoreduplication. A single constant parameter sigma describes the proportion of cells that moves from one to the next class of DNA content after each lapse of time tauE, considered to be the minimum time required for an endocycle. The model calculates the total number of cells and their distribution among eight classes of ploidy level. The dynamic patterns of cell proliferation and ploidy were compared with those obtained experimentally on two contrasting tomato genotypes. The approach developed in this model should allow the future integration of new knowledge concerning the genetic and environmental control of the switch from complete to incomplete cell cycle.

This work proposed a model of tomato growth adapted from the Fishman and Génard model developed to predict carbon and water accumulation in peach fruit. The main adaptations relied on the literature on tomato and mainly concerned: (i) the decrease in cell wall extensibility coefficient during fruit development; (ii) the increase in the membrane reflection coefficient to solute from 0 to 1, which accounted for the switch from symplasmic to apoplasmic phloem unloading, and (iii) the negative influence of the initial fruit weight on the maximum rate of active carbon uptake based on the assumption of higher competition for carbon among cells in large fruits containing more cells. A sensitivity analysis was performed and the model was calibrated and evaluated with satisfaction on 17 experimental datasets obtained under contrasting environmental (temperature, air vapour pressure deficit) and plant (plant fruit load and fruit position) conditions. Then the model was used to analyse the variations in the main fluxes involved in tomato fruit growth and accumulation of carbon in response to virtual carbon and water stresses. The conclusions are that this model, integrating simple biophysical laws, was able to simulate the complex fruit behaviour in response to external or internal factors and thus it may be a powerful tool for managing fruit growth and quality.

The fruit is a hierarchically organized organ composed of cells from different tissues. Its quality, defined by traits such as fruit size and composition, is the result of a complex chain of biological processes. These processes involve exchanges (transpiration, respiration, photosynthesis, phloem and xylem fluxes, and ethylene emission) between the fruit and its environment (atmosphere or plant), tissue differentiation, and cell functioning (division, endoreduplication, expansion, metabolic transformations, and vacuolar storage). In order to progress in our understanding of quality development, it is necessary to analyse the fruit as a system, in which processes interact. In this case, a process-based modelling approach is particularly powerful. Such a modelling approach is proposed to develop a future 'virtual fruit' model. The value of a virtual fruit for agronomists and geneticists is also discussed.

The bronchodilator effect of deep inhalation (DI) may be assessed from the time course of respiratory system resistance (Rrs) and reactance (Xrs) measured by the forced oscillation technique at a single frequency. The aim of the study was to assess the effect of DI in the closed chest rat. Under anesthesia and mechanical ventilation, seven Brown Norway rats were given regular DI (BN-di) and six underwent continuous tidal ventilation (BN) throughout an otherwise similar methacholine (Mch) challenge protocol. Rrs and Xrs were monitored at 20 Hz and apparent respiratory system elastance (Ers) was computed from Xrs. After Mch nebulization, there was a significant increase in Rrs and Ers compared with saline. Ers, but not Rrs, decreased after the DI and BN-di were found to have lower Ers than BN. Thus, DI significantly alters Ers and its response to Mch. Computer simulations suggested reversal of increased viscoelasticity and/or inhomogeneous behavior by the DI in that model.

Although numerous fundamental aspects of development have been uncovered through the study of individual genes and proteins, system-level models are still missing for most developmental processes. The first two cell divisions of Caenorhabditis elegans embryogenesis constitute an ideal test bed for a system-level approach. Early embryogenesis, including processes such as cell division and establishment of cellular polarity, is readily amenable to large-scale functional analysis. A first step toward a system-level understanding is to provide 'first-draft' models both of the molecular assemblies involved and of the functional connections between them. Here we show that such models can be derived from an integrated gene/protein network generated from three different types of functional relationship: protein interaction, expression profiling similarity and phenotypic profiling similarity, as estimated from detailed early embryonic RNA interference phenotypes systematically recorded for hundreds of early embryogenesis genes. The topology of the integrated network suggests that C. elegans early embryogenesis is achieved through coordination of a limited set of molecular machines. We assessed the overall predictive value of such molecular machine models by dynamic localization of ten previously uncharacterized proteins within the living embryo.

Currently available protein-protein interaction (PPI) network or 'interactome' maps, obtained with the yeast two-hybrid (Y2H) assay or by co-affinity purification followed by mass spectrometry (co-AP/MS), only cover a fraction of the complete PPI networks. These partial networks display scale-free topologies--most proteins participate in only a few interactions whereas a few proteins have many interaction partners. Here we analyze whether the scale-free topologies of the partial networks obtained from Y2H assays can be used to accurately infer the topology of complete interactomes. We generated four theoretical interaction networks of different topologies (random, exponential, power law, truncated normal). Partial sampling of these networks resulted in sub-networks with topological characteristics that were virtually indistinguishable from those of currently available Y2H-derived partial interactome maps. We conclude that given the current limited coverage levels, the observed scale-free topology of existing interactome maps cannot be confidently extrapolated to complete interactomes.

RNA interference (RNAi) of target genes is triggered by double-stranded RNAs (dsRNAs) processed by conserved nucleases and accessory factors. To identify the genetic components required for RNAi, we performed a genome-wide screen using an engineered RNAi sensor strain of Caenorhabditis elegans. The RNAi screen identified 90 genes. These included Piwi/PAZ proteins, DEAH helicases, RNA binding/processing factors, chromatin-associated factors, DNA recombination proteins, nuclear import/export factors, and 11 known components of the RNAi machinery. We demonstrate that some of these genes are also required for germline and somatic transgene silencing. Moreover, the physical interactions among these potential RNAi factors suggest links to other RNA-dependent gene regulatory pathways.

Background and Aims: To better understand the regulation of fruit growth in response to environmental factors, the effects of temperature and plant fruit load on cell number, cell size and DNA endoreduplication were analysed., Methods: Plants were grown at 20/20 degrees C, 25/25 degrees C and 25/20 degrees C day/night temperatures, and inflorescences were pruned to two ('2F') or five ('5F') flowers., Key Results and Conclusions: Despite a lower fruit growth rate at 20/20 degrees C, temperature did not affect final fruit size because of the compensation between cell number and size. The higher cell number at 20/20 degrees C (9.0 x 10(6) against 7.9 x 10(6) at 25/25 degrees C and 7.7 x 10(6) at 25/20 degrees C) resulted from an extended period of cell division, and the smaller cell size was due to a shorter period of expansion rather than a lower expansion rate. By contrast, the lower fruit growth rate and size of 5F fruits compared with 2F fruits resulted from the slow down of cell expansion, whereas the number of cells was hardly affected in the proximal fruit. However, within the inflorescence the decreasing gradient of fruit size from proximal to distal fruits was due to a decrease in cell number with similar cell size. Fruit size variations within each treatment were always positively correlated to variations in cell number, but not in cell size. Negative correlations between cell size and cell number suggested that cells of tomato pericarp can be seen as a population of competing sinks. Mean ploidy was slightly delayed and reduced in 5F fruits compared with 2F fruits. It was highest at 25/25 degrees C and lowest at 25/20 degrees C. Treatments did not affect ploidy and cell size in similar ways, but within each treatment, positive correlations existed between mean ploidy and cell size, though significant only in the 2F-25/20 treatment.

The advent of systems biology necessitates the cloning of nearly entire sets of protein-encoding open reading frames (ORFs), or ORFeomes, to allow functional studies of the corresponding proteomes. Here, we describe the generation of a first version of the human ORFeome using a newly improved Gateway recombinational cloning approach. Using the Mammalian Gene Collection (MGC) resource as a starting point, we report the successful cloning of 8076 human ORFs, representing at least 7263 human genes, as mini-pools of PCR-amplified products. These were assembled into the human ORFeome version 1.1 (hORFeome v1.1) collection. After assessing the overall quality of this version, we describe the use of hORFeome v1.1 for heterologous protein expression in two different expression systems at proteome scale. The hORFeome v1.1 represents a central resource for the cloning of large sets of human ORFs in various settings for functional proteomics of many types, and will serve as the foundation for subsequent improved versions of the human ORFeome.

The bacteria of the Brucella genus are responsible for a worldwide zoonosis called brucellosis. They belong to the alpha-proteobacteria group, as many other bacteria that live in close association with a eukaryotic host. Importantly, the Brucellae are mainly intracellular pathogens, and the molecular mechanisms of their virulence are still poorly understood. Using the complete genome sequence of Brucella melitensis, we generated a database of protein-coding open reading frames (ORFs) and constructed an ORFeome library of 3091 Gateway Entry clones, each containing a defined ORF. This first version of the Brucella ORFeome (v1.1) provides the coding sequences in a user-friendly format amenable to high-throughput functional genomic and proteomic experiments, as the ORFs are conveniently transferable from the Entry clones to various Expression vectors by recombinational cloning. The cloning of the Brucella ORFeome v1.1 should help to provide a better understanding of the molecular mechanisms of virulence, including the identification of bacterial protein-protein interactions, but also interactions between bacterial effectors and their host's targets.

Aim: To measure the change in blood-pressure control in two populations of patients with severe high blood pressure between 1999 and 2003., Method: The French National Healthcare Fund performed two observational, cross-sectional surveys on the medical management of high blood pressure, the first in 1999 and the second in 2003. Each survey enrolled patients aged between 20 and 80 years old who filed a first-time request for exemption from co-payments for this long-term disorder. Study data was collected by the health fund's salaried physician advisors who directly examined the patients and from information gathered from each patient's attending physician. Adequate blood pressure control was defined according to the guidelines set out by the ANAES in 1997 and determined by calculating the average of the last three blood pressure figures recorded by the patient's attending physician (systolic [SBP] and diastolic [DBP] arterial blood pressures)., Results: 10,665 patients were enrolled in the 1999 survey and 2,584 were enrolled in the 2003 survey. The average age was 63.1 +/- 0.2 years in 1999 and 64.4 +/- 0.4 years in 2003. The proportion of diabetic patients in the two surveys remained stable: 27.5% +/- 1.0 in 1999 and 28.4% +/- 1.7 in 2003. Between 1999 and 2003, the percentage of patient who were treated for hypercholesterolemia increased from 44.0% +/- 1.1 to 54.3% +/- 1.9. Between 1999 and 2003, the proportion of patients who were considered well-controlled (SBP and DBP < 140/90 mmHg) or, if older than 60 years with isolated systolic high blood pressure (DBP < 90 mmHg and SBP < or = 160 mmHg) increased almost 5% points, going from 40.8% +/- 1.1 in 1999 to 45.5% +/- 1.9 in 2003. In diabetic patients, in whom the cut-off threshold is 130/85 mmHg, 6.7% +/- 1.1 were considered well-controlled in 1999 while 5.4% +/- 1.7 were deemed well-controlled in 2003. The proportion of well-controlled patients in the sub-group of hypertensive patients with renal failure (cut-off thresholds: 125/75 mmHg) remained relatively stable between 1999 and 2003: 5.2% [2.5; 7.9] versus 2.8% [0.5; 8.6]., Conclusion: There was a significant increase in the proportion of well-controlled hypertensive patients between 1999 and 2003. This increase occurred at the same time as a number of initiatives (scientific societies, federal government as well as the National Health Fund) intended to sensitize physicians to the need to obtain adequate blood pressure control.

In apparently scale-free protein-protein interaction networks, or 'interactome' networks, most proteins interact with few partners, whereas a small but significant proportion of proteins, the 'hubs', interact with many partners. Both biological and non-biological scale-free networks are particularly resistant to random node removal but are extremely sensitive to the targeted removal of hubs. A link between the potential scale-free topology of interactome networks and genetic robustness seems to exist, because knockouts of yeast genes encoding hubs are approximately threefold more likely to confer lethality than those of non-hubs. Here we investigate how hubs might contribute to robustness and other cellular properties for protein-protein interactions dynamically regulated both in time and in space. We uncovered two types of hub: 'party' hubs, which interact with most of their partners simultaneously, and 'date' hubs, which bind their different partners at different times or locations. Both in silico studies of network connectivity and genetic interactions described in vivo support a model of organized modularity in which date hubs organize the proteome, connecting biological processes--or modules--to each other, whereas party hubs function inside modules.

Proteins function mainly through interactions, especially with DNA and other proteins. While some large-scale interaction networks are now available for a number of model organisms, their experimental generation remains difficult. Consequently, interolog mapping--the transfer of interaction annotation from one organism to another using comparative genomics--is of significant value. Here we quantitatively assess the degree to which interologs can be reliably transferred between species as a function of the sequence similarity of the corresponding interacting proteins. Using interaction information from Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, and Helicobacter pylori, we find that protein-protein interactions can be transferred when a pair of proteins has a joint sequence identity >80% or a joint E-value <10(-70). (These "joint" quantities are the geometric means of the identities or E-values for the two pairs of interacting proteins.) We generalize our interolog analysis to protein-DNA binding, finding such interactions are conserved at specific thresholds between 30% and 60% sequence identity depending on the protein family. Furthermore, we introduce the concept of a "regulog"--a conserved regulatory relationship between proteins across different species. We map interologs and regulogs from yeast to a number of genomes with limited experimental annotation (e.g., Arabidopsis thaliana) and make these available through an online database at http://interolog.gersteinlab.org. Specifically, we are able to transfer approximately 90,000 potential protein-protein interactions to the worm. We test a number of these in two-hybrid experiments and are able to verify 45 overlaps, which we show to be statistically significant., (Copyright 2004 Cold Spring Harbor Laboratory Press)

To initiate a system-level analysis of C. elegans DAF-7/TGF-beta signaling, we combined interactome mapping with single and double genetic perturbations. Yeast two-hybrid (Y2H) screens starting with known DAF-7/TGF-beta pathway components defined a network of 71 interactions among 59 proteins. Coaffinity purification (co-AP) assays in mammalian cells confirmed the overall quality of this network. Systematic perturbations of the network using RNAi, both in wild-type and daf-7/TGF-beta pathway mutant animals, identified nine DAF-7/TGF-beta signaling modifiers, seven of which are conserved in humans. We show that one of these has functional homology to human SNO/SKI oncoproteins and that mutations at the corresponding genetic locus daf-5 confer defects in DAF-7/TGF-beta signaling. Our results reveal substantial molecular complexity in DAF-7/TGF-beta signal transduction. Integrating interactome maps with systematic genetic perturbations may be useful for developing a systems biology approach to this and other signaling modules.

To initiate studies on how protein-protein interaction (or "interactome") networks relate to multicellular functions, we have mapped a large fraction of the Caenorhabditis elegans interactome network. Starting with a subset of metazoan-specific proteins, more than 4000 interactions were identified from high-throughput, yeast two-hybrid (HT=Y2H) screens. Independent coaffinity purification assays experimentally validated the overall quality of this Y2H data set. Together with already described Y2H interactions and interologs predicted in silico, the current version of the Worm Interactome (WI5) map contains approximately 5500 interactions. Topological and biological features of this interactome network, as well as its integration with phenome and transcriptome data sets, lead to numerous biological hypotheses.

This work investigated the link between genetic and developmental controls of fruit size and composition. On two isogenic lines (CF12-C and CF14-L), differing by fruit weight and sugar content quantitative trait loci (QTLs) identified previously, basal and tip fruits were characterized at anthesis and at maturity through their growth, dry matter and sugar content, number and size of cells and nuclei DNA content. The influence of competition was assessed by removing either basal or tip ovaries at anthesis. On an intact inflorescence, CF12-C fruits grew less than CF14-L fruits, with 1.67 fewer cell layers and similar cell size, suggesting that genes controlling cell division may be responsible for this fruit size variation. Truss thinning masked the QTL effect on fruit size, mainly by reducing the difference in cell number between the two lines and by promoting cell expansion in tip fruits, so that fruit growth was similar at both positions and for both lines. Thus, in these lines, cell number exerts a control on final fruit size only when there is competition among fruits. Different responses of basal and tip fruits after flower removal suggested that this treatment induced changes in hormonal relationships within the truss. No fixed relationship between DNA endoreduplication and cell size was found, as while cell size and dry matter and sugar contents differed with tomato lines, fruit position and truss size, endoreduplication patterns were the same. CF12-C fruits had a higher dry matter (+0.3% of fresh weight) and carbohydrates (+8% of dry matter) content than CF14-L fruits. The percentage dry matter was independent of truss size but decreased slightly from basal to tip fruits.

Changes in cell number during the early period of tomato fruit development were analysed by means of a deterministic model of cell multiplication. The period commenced at the seed stage with one theoretical cell undergoing intensive cell division, and ended when the cell number became nearly constant. The model takes into consideration the proliferative activity of the fruit cell population which, a few days before flower anthesis, begins to decrease progressively after each mitotic cycle. Model parameters, namely the time at which proliferative activity diminishes, its rate of decrease and the length of the cell cycle, were estimated by fitting the model to observed cell population dynamics in tomato fruits growing in three different positions on the truss. It is hypothesized that the molecular mechanism responsible for the cessation of mitosis in growing fruits is associated with shortening of telomeric ends of nuclear DNA, as suggested previously for other growing cell populations.

To verify the genome annotation and to create a resource to functionally characterize the proteome, we attempted to Gateway-clone all predicted protein-encoding open reading frames (ORFs), or the 'ORFeome,' of Caenorhabditis elegans. We successfully cloned approximately 12,000 ORFs (ORFeome 1.1), of which roughly 4,000 correspond to genes that are untouched by any cDNA or expressed-sequence tag (EST). More than 50% of predicted genes needed corrections in their intron-exon structures. Notably, approximately 11,000 C. elegans proteins can now be expressed under many conditions and characterized using various high-throughput strategies, including large-scale interactome mapping. We suggest that similar ORFeome projects will be valuable for other organisms, including humans.

To evaluate the relevance of a simple carbon balance model (Seginer et al., 1994, Scientia Horticulturae 60: 55-80) in source-limiting conditions, the dynamics of growth, respiration and carbohydrate reserves of tomato plants were observed in prolonged darkness. Four days prior to the experiments, plants were exposed to high or low light levels and CO(2) concentrations. The concentration of carbohydrates in vegetative organs was 30-50 % lower in plants that were exposed to low carbon assimilation conditions compared with those exposed to high carbon assimilation conditions. During prolonged darkness, plants with low carbohydrate reserves exhibited a lower whole-plant respiration rate, which decreased rapidly to almost zero after 24 h, and carbohydrate pools were almost exhausted in leaves, roots and flowers. In plants with high carbohydrate reserves, the whole-plant respiration rate was maintained for a longer period and carbohydrates remained available for at least 48 h in leaves and flowers. In contrast, fruits maintained fairly stable and identical concentrations of carbohydrates and the reduction in their rate of expansion was moderate irrespective of the pre-treatment carbon assimilation conditions. The time-course of asparagine and glutamine concentrations showed the occurrence of carbon stress in leaves and flowers. Estimation of source and sink activities indicated that even after low carbon assimilation, vegetative organs contained enough carbohydrates to support fruit growth provided their own growth stopped. The time of exhaustion of these carbohydrates corresponded grossly to the maintenance stage simulated by the model proposed by Seginer et al. (1994), thus validating the use of such a model for optimizing plant growth.

By integrating functional genomic and proteomic mapping approaches, biological hypotheses should be formulated with increasing levels of confidence. For example, yeast interactome and transcriptome data can be correlated in biologically meaningful ways. Here, we combine interactome mapping data generated for a multicellular organism with data from both large-scale phenotypic analysis ("phenome mapping") and transcriptome profiling. First, we generated a two-hybrid interactome map of the Caenorhabditis elegans germline by using 600 transcripts enriched in this tissue. We compared this map to a phenome map of the germline obtained by RNA interference (RNAi) and to a transcriptome map obtained by clustering worm genes across 553 expression profiling experiments. In this dataset, we find that essential proteins have a tendency to interact with each other, that pairs of genes encoding interacting proteins tend to exhibit similar expression profiles, and that, for approximately 24% of germline interactions, both partners show overlapping embryonic lethal or high incidence of males RNAi phenotypes and similar expression profiles. We propose that these interactions are most likely to be relevant to germline biology. Similar integration of interactome, phenome, and transcriptome data should be possible for other biological processes in the nematode and for other organisms, including humans.

Unlabelled: The mammary gland is an organ under complex hormonal and developmental regulation. Among the mediators involved in this process are polypeptides, steroid hormones and certain lipids and their metabolites. The fatty acid content of the breast adipocytes is investigated because of the importance of their role in the proliferation and differentiation of the breast epithelial cells., Materials and Methods: To address the hypothesis that polyunsaturated acid might be a risk factor for breast cancer, we examined the association between arachidonic acid (20:4n-6) and menopausal status in the women using lipid extraction from isolated human adipocytes and CD36 detection on a FACS flow cytometer., Results: Compared with the related trigycerides, phospholipids in postmenopausal women contained a higher proportion of polyunsaturated fatty acids like arachidonic acid, a precursor of eicosanoids, required for many physiological roles in reproduction and implicated in the proliferation of several cell types. In order to determine the mechanisms explaining the difference in the fatty acid and long-chain fatty acid composition, we have studied the relationship between CD 36 protein and menopausal status. Menopause in women seemed to be associated with a diminuation of the amount of CD 36 protein of adipocytes (48.82 +/- 3.54% versus 20.01 +/- 0.04%) and also associated with the increased percentage of arachidonic acid observed in postmenopausal women. Our data and others led us to conclude that the variation in the composition of fatty acids can act directly on the phenomena of growth and differentiation of mammary epithelial cells and the variation in CD 36 may act either on the transport of fatty acids or on the transduction of the signal responsible for the stimulation of enzymes catalysing the conversion of arachidonic acid into different metabolites. These two phenomena could influence the risk of breast cancer in pre- and postmenopausal women.

Thrombospondin-1 (TSP1) is a Mr 450,000 extracellular matrix glycoprotein that modulates tumor growth, angiogenesis, and metastasis. Of the five structurally different TSPs described to date, only TSP2 is similar to TSP1 in terms of its molecular architecture, and TSP2 also modulates angiogenesis. Angiogenesis plays a relevant role in the biological aggressiveness of breast cancer, and TSP1 is present in the tumor stroma (termed desmoplasia) of invasive human breast ductal carcinoma not otherwise specified (NOS). The present study was designed to identify and quantify TSP1 and TSP2 mRNAs in normal, benign, and neoplastic human breast tissues using the reverse transcriptase PCR technique. We found that TSP2, like TSP1, was expressed in human breast tissues, and that TSP1 and TSP2 mRNA expression in invasive breast carcinoma NOS was significantly increased compared to that observed in normal and benign tissues. The expression of TSP1 and TSP2 in invasive breast ductal carcinoma NOS did not significantly correlate with any of the prognostic factors studied (tumor size, lymph node status, morphology, and hormone receptor status). However, when our study population was divided according to the quantity of tumor stroma, TSP1 (and possibly TSP2) mRNA expression and microvessel counts in desmoplastic-rich stroma of breast carcinoma NOS were significantly increased compared to those observed in desmoplastic-poor stromata.

We investigated the effects of a short period of water stress on monoterpene emissions from Quercus ilex, a common oak species of the Mediterranean vegetation and a strong emitter of monoterpenes. The experiment was carried out on two young saplings with a branch enclosure system under semi-controlled conditions. Under unstressed conditions, small qualitative (cis-β-ocimene, trans-β-ocimene, β-caryophyllene and 1,8-cineol) and large quantitative (as much as 40% for the main compounds emitted) differences were observed between the two apparently similar trees. Nevertheless these differences did not affect the short-and long-term responses to temperature and water stress. Daily courses of emissions and gas exchanges were similar before and after the stress. During the most severe stress, emissions were reduced by a factor of two orders of magnitude and the log-linear relationship between emissions and temperature no longer existed. Photosynthesis and transpiration rates decreased as soon as the soil started to dry, whereas monoterpene emissions slightly increased for few days and then dropped when the daily CO 2 balance approached zero. We concluded that under water stress monoterpene emissions were highly limited by monoterpene synthesis resulting from a lack of carbon substrate and/or ATP. After rewatering, both emissions and gas exchanges recovered immediately, but to a level lower than the pre-stress level. These results have many implications for monoterpene emission modelling in the Mediterranean area, since the dry period generally extends from May to August. If our results are confirmed by field experiments, water stress could lead to a large overestimation of the emissions under summer conditions, when the algorithms based on light and temperature would give high emission rates.

Localization and characterization of fruit set in winter tomato crops was investigated to determine the main internal and external controlling factors and to establish a quantitative relationship between fruit set and competition for assimilates. Individual fruit growth and development was assessed on a beef tomato cultivar during the reproductive period (first nine inflorescences). A non-destructive photograph technique was used to measure fruit growth from very early stages of their development and then calliper measurements were made on big fruits. From these measurements we determined the precise developmental stage at which fruit growth stopped. Fruit potential growth, which is defined as the growth achieved in non-limiting conditions for assimilate supply, was also assessed by this method on plants thinned to one flower per inflorescence. The latter was used to calculate the ratio between actual and potential growth, which was found to be a good index of the competition for assimilates. Time lags of fruit set were observed mainly on distal organs. When more than three flowers were left on each inflorescence, distal organs developed at the same time as proximal organs of the following inflorescence. Consequently they were submitted to a double competition within one inflorescence and among inflorescences. It was shown that, what is commonly named 'fruit set failure', is not an irreversible death of the organ and that a small fruit could resume growth after a delay of several weeks as soon as the first fruits ripened and thus ceased to compete for assimilates. In that case proximal fruits resumed growth before distal ones. The delayed fruits contained only few seeds but a germination test confirmed that fertilization took place before fruit set failed. Competition for assimilates was calculated during plant development by the ratio between actual and potential fruit growth. Potential growth of proximal fruits was strongly dependent on the position of the inflorescence on the stem, whereas potential growth of distal fruits was lower than or equal to that of proximal fruits of the same inflorescence and it was independent on the inflorescence position. We took into account both inflorescence and fruit positions to establish a quantitative relationship between fruit set of individual inflorescences and the ratio between actual and potential fruit growth.

Thrombospondin (TSP) is a 450 kDa extracellular matrix glycoprotein expressed in normal, hyperplastic, and neoplastic human breast. In this study, the patterns of expression of TSP were determined during development of the human fetal mammary gland between the 15th and the 39th week of gestation. Using immunohistochemistry, TSP is found in the dense mesenchyme immediately adjacent to the mammary bud, and at the membrane of budding epithelial cells invading the surrounding mesenchyme. As formation of the ductal tree system occurs, TSP is deposited at the myoepithelial-stromal junction of mammary ducts. Such an immunolocalization of TSP in buds and ducts of the fetal mammary gland has been confirmed at the mRNA level using in situ hybridization. Presence of TSP transcripts in nascent breast tissue has been also demonstrated by polymerase chain reaction assay. Comparison of TSP immunolocalization with that of two known TSP cell surface receptors, CD36 and CD51, reveals no codistribution of TSP with these receptors during mammary gland development. As opposed to TSP, CD36 is strongly expressed at the membrane of preadipocytes present in the fat pad tissue, but absent from budding epithelial cells. CD51 is only weakly expressed by malpighian epithelial cells and does not colocalize with TSP. In lactating ducts of a newborn, TSP disappears from the myoepithelial-stromal junction of ducts and is synthesized at the apices of secretory epithelial cells of lactating ducts together with CD36. In conclusion, our findings support the existence of an important role for TSP during development of the human fetal mammary gland.(ABSTRACT TRUNCATED AT 250 WORDS)