Proteome analysis of yeast response to various nutrient limitations

We compared the response of Saccharomyces cerevisiae to carbon (glucose) and nitrogen (ammonia) limitation in chemostat cultivation at the proteome level. Protein levels were differentially quantified using unlabeled and (15)N metabolically labeled yeast cultures. A total of 928 proteins covering a wide range of isoelectric points, molecular weights and subcellular localizations were identified. Stringent statistical analysis identified 51 proteins upregulated in response to glucose limitation and 51 upregulated in response to ammonia limitation. Under glucose limitation, typical glucose-repressed genes encoding proteins involved in alternative carbon source utilization, fatty acids β-oxidation and oxidative phosphorylation displayed an increased protein level. Proteins upregulated in response to nitrogen limitation were mostly involved in scavenging of alternative nitrogen sources and protein degradation. Comparison of transcript and protein levels clearly showed that upregulation in response to glucose limitation was mainly transcriptionally controlled, whereas upregulation in response to nitrogen limitation was essentially controlled at the post-transcriptional level by increased translational efficiency and/or decreased protein degradation. These observations underline the need for multilevel analysis in yeast systems biology

Mosaicism for combined tetrasomy of chromosomes 8 and 18 in a dysmorphic child: A result of failed tetraploidy correction?

<p>Abstract</p> <p>Background</p> <p>Mosaic whole-chromosome tetrasomy has not previously been described as a cause of fetal malformations.</p> <p>Case presentation</p> <p>In a markedly dysmorphic child with heart malformations and developmental delay, CGH analysis of newborn blood DNA suggested a 50% dose increase of chromosomes 8 and 18, despite a normal standard karyotype investigation. Subsequent FISH analysis revealed leukocytes with four chromosomes 8 and four chromosomes 18. The child's phenotype had resemblance to both mosaic trisomy 8 and mosaic trisomy 18. The double tetrasomy was caused by mitotic malsegregation of all four chromatids of both chromosome pairs. A possible origin of such an error is incomplete correction of a tetraploid state resulting from failed cytokinesis or mitotic slippage during early embryonic development.</p> <p>Conclusion</p> <p>This unique case suggests that embryonic cells may have a mechanism for tetraploidy correction that involves mitotic pairing of homologous chromosomes.</p

The Effect of a DNA Damaging Agent on Embryonic Cell Cycles of the Cnidarian Hydractinia echinata

The onset of gastrulation at the Mid-Blastula Transition can accompany profound changes in embryonic cell cycles including the introduction of gap phases and the transition from maternal to zygotic control. Studies in Xenopus and Drosophila embryos have also found that cell cycles respond to DNA damage differently before and after MBT (or its equivalent, MZT, in Drosophila). DNA checkpoints are absent in Xenopus cleavage cycles but are acquired during MBT. Drosophila cleavage nuclei enter an abortive mitosis in the presence of DNA damage whereas post-MZT cells delay the entry into mitosis. Despite attributes that render them workhorses of embryonic cell cycle studies, Xenopus and Drosophila are hardly representative of diverse animal forms that exist. To investigate developmental changes in DNA damage responses in a distant phylum, I studied the effect of an alkylating agent, Methyl Methanesulfonate (MMS), on embryos of Hydractinia echinata. Hydractinia embryos are found to differ from Xenopus embryos in the ability to respond to a DNA damaging agent in early cleavage but are similar to Xenopus and Drosophila embryos in acquiring stronger DNA damage responses and greater resistance to killing by MMS after the onset of gastrulation. This represents the first study of DNA damage responses in the phylum Cnidaria

The vertebrate phylotypic stage and an early bilaterian-related stage in mouse embryogenesis defined by genomic information

BACKGROUND: Embryos of taxonomically different vertebrates are thought to pass through a stage in which they resemble one another morphologically. This "vertebrate phylotypic stage" may represent the basic vertebrate body plan that was established in the common ancestor of vertebrates. However, much controversy remains about when the phylotypic stage appears, and whether it even exists. To overcome the limitations of studies based on morphological comparison, we explored a comprehensive quantitative method for defining the constrained stage using expressed sequence tag (EST) data, gene ontologies (GO), and available genomes of various animals. If strong developmental constraints occur during the phylotypic stage of vertebrate embryos, then genes conserved among vertebrates would be highly expressed at this stage. RESULTS: We established a novel method for evaluating the ancestral nature of mouse embryonic stages that does not depend on comparative morphology. The numerical "ancestor index" revealed that the mouse indeed has a highly conserved embryonic period at embryonic day 8.0–8.5, the time of appearance of the pharyngeal arch and somites. During this period, the mouse prominently expresses GO-determined developmental genes shared among vertebrates. Similar analyses revealed the existence of a bilaterian-related period, during which GO-determined developmental genes shared among bilaterians are markedly expressed at the cleavage-to-gastrulation period. The genes associated with the phylotypic stage identified by our method are essential in embryogenesis. CONCLUSION: Our results demonstrate that the mid-embryonic stage of the mouse is indeed highly constrained, supporting the existence of the phylotypic stage. Furthermore, this candidate stage is preceded by a putative bilaterian ancestor-related period. These results not only support the developmental hourglass model, but also highlight the hierarchical aspect of embryogenesis proposed by von Baer. Identification of conserved stages and tissues by this method in various animals would be a powerful tool to examine the phylotypic stage hypothesis, and to understand which kinds of developmental events and gene sets are evolutionarily constrained and how they limit the possible variations of animal basic body plans

Unexpected Role of α-Fetoprotein in Spermatogenesis

BACKGROUND: Heat shock severely affects sperm production (spermatogenesis) and results in a rapid loss of haploid germ cells, or in other words, sperm formation (spermiogenesis) is inhibited. However, the mechanisms behind the effects of heat shock on spermatogenesis are obscure. METHODOLOGY/PRINCIPAL FINDINGS: To identify the inhibitory factor of spermiogenesis, experimental cryptorchid (EC) mice were used in this study. Here we show that α-fetoprotein (AFP) is specifically expressed in the testes of EC mice by proteome analysis. AFP was also specifically localized spermatocytes by immunohistochemical analysis and was secreted into the circulation system of EC mice by immunoblot analysis. Since spermatogenesis of an advanced mammal cannot be reproduced with in vitro, we performed the microinjection of AFP into the seminiferous tubules of normal mice to determine whether AFP inhibits spermiogenesis in vivo. AFP was directly responsible for the block in spermiogenesis of normal mice. To investigate whether AFP inhibits cell differentiation in other models, using EC mice we performed a partial hepatectomy (PH) that triggers a rapid regenerative response in the remnant liver tissue. We also found that liver regeneration is inhibited in EC mice with PH. The result suggests that AFP released into the blood of EC mice regulates liver regeneration by inhibiting the cell division of hepatocytes. CONCLUSIONS/SIGNIFICANCE: AFP is a well-known cancer-specific marker, but AFP has no known function in healthy human beings. Our findings indicate that AFP expressed under EC conditions plays a role as a regulatory factor in spermatogenesis and in hepatic generation

A Low Protein Diet Increases the Hypoxic Tolerance in Drosophila

Dietary restriction is well known to increase the life span of a variety of organisms from yeast to mammals, but the relationships between nutrition and the hypoxic tolerance have not yet been considered. Hypoxia is a major cause of cell death in myocardial infarction and stroke. Here we forced hypoxia-related death by exposing one-day-old male Drosophila to chronic hypoxia (5% O(2)) and analysed their survival. Chronic hypoxia reduced the average life span from 33.6 days to 6.3 days when flies were fed on a rich diet. A demographic analysis indicated that chronic hypoxia increased the slope of the mortality trajectory and not the short-term risk of death. Dietary restriction produced by food dilution, by yeast restriction, or by amino acid restriction partially reversed the deleterious action of hypoxia. It increased the life span of hypoxic flies up to seven days, which represented about 25% of the life time of an hypoxic fly. Maximum survival of hypoxic flies required only dietary sucrose, and it was insensitive to drugs such as rapamycin and resveratrol, which increase longevity of normoxic animals. The results thus uncover a new link between protein nutrition, nutrient signalling, and resistance to hypoxic stresses

A DIGE study on the effects of salbutamol on the rat muscle proteome - an exemplar of best practice for data sharing in proteomics

BACKGROUND: Proteomic techniques allow researchers to perform detailed analyses of cellular states and many studies are published each year, which highlight large numbers of proteins quantified in different samples. However, currently few data sets make it into public databases with sufficient metadata to allow other groups to verify findings, perform data mining or integrate different data sets. The Proteomics Standards Initiative has released a series of "Minimum Information About a Proteomics Experiment" guideline documents (MIAPE modules) and accompanying data exchange formats. This article focuses on proteomic studies based on gel electrophoresis and demonstrates how the corresponding MIAPE modules can be fulfilled and data deposited in public databases, using a new experimental data set as an example. FINDINGS: We have performed a study of the effects of an anabolic agent (salbutamol) at two different time points on the protein complement of rat skeletal muscle cells, quantified by difference gel electrophoresis. In the DIGE study, a total of 31 non-redundant proteins were identified as being potentially modulated at 24 h post treatment and 110 non redundant proteins at 96 h post-treatment. Several categories of function have been highlighted as strongly enriched, providing candidate proteins for further study. We also use the study as an example of best practice for data deposition. CONCLUSIONS: We have deposited all data sets from this study in public databases for further analysis by the community. We also describe more generally how gel-based protein identification data sets can now be deposited in the PRoteomics IDEntifications database (PRIDE), using a new software tool, the PRIDESpotMapper, which we developed to work in conjunction with the PRIDE Converter application. We also demonstrate how the ProteoRed MIAPE generator tool can be used to create and share a complete and compliant set of MIAPE reports for this experiment and others

Malaria Parasite Invasion of the Mosquito Salivary Gland Requires Interaction between the Plasmodium TRAP and the Anopheles Saglin Proteins

SM1 is a twelve-amino-acid peptide that binds tightly to the Anopheles salivary gland and inhibits its invasion by Plasmodium sporozoites. By use of UV-crosslinking experiments between the peptide and its salivary gland target protein, we have identified the Anopheles salivary protein, saglin, as the receptor for SM1. Furthermore, by use of an anti-SM1 antibody, we have determined that the peptide is a mimotope of the Plasmodium sporozoite Thrombospondin Related Anonymous Protein (TRAP). TRAP binds to saglin with high specificity. Point mutations in TRAP's binding domain A abrogate binding, and binding is competed for by the SM1 peptide. Importantly, in vivo down-regulation of saglin expression results in strong inhibition of salivary gland invasion. Together, the results suggest that saglin/TRAP interaction is crucial for salivary gland invasion by Plasmodium sporozoites

Spire, an Actin Nucleation Factor, Regulates Cell Division during Drosophila Heart Development

The Drosophila dorsal vessel is a beneficial model system for studying the regulation of early heart development. Spire (Spir), an actin-nucleation factor, regulates actin dynamics in many developmental processes, such as cell shape determination, intracellular transport, and locomotion. Through protein expression pattern analysis, we demonstrate that the absence of spir function affects cell division in Myocyte enhancer factor 2-, Tinman (Tin)-, Even-skipped- and Seven up (Svp)-positive heart cells. In addition, genetic interaction analysis shows that spir functionally interacts with Dorsocross, tin, and pannier to properly specify the cardiac fate. Furthermore, through visualization of double heterozygous embryos, we determines that spir cooperates with CycA for heart cell specification and division. Finally, when comparing the spir mutant phenotype with that of a CycA mutant, the results suggest that most Svp-positive progenitors in spir mutant embryos cannot undergo full cell division at cell cycle 15, and that Tin-positive progenitors are arrested at cell cycle 16 as double-nucleated cells. We conclude that Spir plays a crucial role in controlling dorsal vessel formation and has a function in cell division during heart tube morphogenesis

The Cell Cycle Time of CD8+ T Cells Responding In Vivo Is Controlled by the Type of Antigenic Stimulus

A hallmark of cells comprising the mammalian adaptive immune system is the requirement for these rare naïve T (and B) lymphocytes directed to a specific microorganism to undergo proliferative expansion upon first encounter with this antigen. In the case of naïve CD8+ T cells the ability of these rare quiescent lymphocytes to rapidly activate and expand into effector T cells in numbers sufficient to control viral and certain bacterial infections can be essential for survival. In this report we examined the activation, cell cycle time and initial proliferative response of naïve murine CD8+ T cells responding in vivo to Influenza and Vaccinia virus infection or vaccination with viral antigens. Remarkably, we observed that CD8+ T cells could divide and proliferate with an initial cell division time of as short as 2 hours. The initial cell cycle time of responding CD8+ T cells is not fixed but is controlled by the antigenic stimulus provided by the APC in vivo. Initial cell cycle time influences the rate of T cell expansion and the numbers of effector T cells subsequently accumulating at the site of infection. The T cell cycle time varies with duration of the G1 phase of the cell cycle. The duration of G1 is inversely correlated with the phosphorylation state of the retinoblastoma (Rb) protein in the responding T cells. The implication of these findings for the development of adaptive immune responses and the regulation of cell cycle in higher eukaryotic cells is discussed