A comprehensive molecular interaction map of the budding yeast cell cycle.

With the accumulation of data on complex molecular machineries coordinating cell-cycle dynamics, coupled with its central function in disease patho-physiologies, it is becoming increasingly important to collate the disparate knowledge sources into a comprehensive molecular network amenable to systems-level analyses. In this work, we present a comprehensive map of the budding yeast cell-cycle, curating reactions from ∼600 original papers. Toward leveraging the map as a framework to explore the underlying network architecture, we abstract the molecular components into three planes--signaling, cell-cycle core and structural planes. The planar view together with topological analyses facilitates network-centric identification of functions and control mechanisms. Further, we perform a comparative motif analysis to identify around 194 motifs including feed-forward, mutual inhibitory and feedback mechanisms contributing to cell-cycle robustness. We envisage the open access, comprehensive cell-cycle map to open roads toward community-based deeper understanding of cell-cycle dynamics.

In vivo robustness analysis of cell division cycle genes in Saccharomyces cerevisiae.

Intracellular biochemical parameters, such as the expression level of gene products, are considered to be optimized so that a biological system, including the parameters, works effectively. Those parameters should have some permissible range so that the systems have robustness against perturbations, such as noise in gene expression. However, little is known about the permissible range in real cells because there has been no experimental technique to test it. In this study, we developed a genetic screening method, named "genetic tug-of-war" (gTOW) that evaluates upper limit copy numbers of genes in a model eukaryote Saccharomyces cerevisiae, and we applied it for 30 cell-cycle related genes (CDC genes). The experiment provided unique quantitative data that could be used to argue the system-level properties of the cell cycle such as robustness and fragility. The data were used to evaluate the current computational model, and refinements to the model were suggested.

Insights into gene expression profiles induced by Socs3 depletion in keratinocytes.

Specific deletion of suppressor of cytokine signaling 3 (Socs3) in keratinocytes can cause severe skin inflammation with infiltration of immune cells. The molecular mechanisms and key regulatory pathways involved in these processes remain elusive. To investigate the role of Socs3 in keratinocytes, we generated and analyzed global RNA-Seq profiles from Socs3 conditional knockout (cKO) mice of two different ages (2 and 10 weeks). Over 400 genes were significantly regulated at both time points. Samples from 2-week-old mice exhibited down-regulation of genes involved in keratin-related functions and up-regulation of genes involved in lipid metabolism. At week 10, multiple chemokine and cytokine genes were up-regulated. Functional annotation revealed that the genes differentially expressed in the 2-week-old mice play roles in keratinization, keratinocyte differentiation, and epidermal cell differentiation. By contrast, differentially expressed genes in the 10-week-old animals are involved in acute immune-related functions. A group of activator protein-1-related genes were highly up-regulated in Socs3 cKO mice of both ages. This observation was validated using qRT-PCR by SOCS3-depleted human keratinocyte-derived HaCaT cells. Our results suggest that, in addition to participating in immune-mediated pathways, SOCS3 also plays important roles in skin barrier homeostasis.

Large deletions in mitochondrial DNA in radiation-associated human thyroid tumors.

Paired DNA samples of tumor and normal thyroid tissue from adult patients possibly exposed to radioactive Chernobyl fallout [11 cases of papillary thyroid carcinoma (PTC) and 6 follicular adenomas] and from control samples (9 PTC occurring in Japanese patients) were examined for the relative mitochondrial DNA (mtDNA) content, prevalence and level of common deletion (CD), and large-scale deletions in mtDNA. Elevated relative mtDNA content as estimated by real-time PCR was found in tumor tissue in most cases, but no significant correlation with the level of radioiodine contamination of patients' residency nor with clinicopathological data were found. CD was detected in every DNA specimen from all types of tissue regardless of the presence of oxyphillic cell changes. Elevated level of the CD was predominantly found in tumor tissue of the radiation-associated group but not in sporadic PTC. No correlation was noted with clinicopathological parameters, radioiodine contamination, and relative mtDNA content. The quantity of large-scale deletions in mtDNA was elevated in most tumor tissues, especially in the radiation-associated group and tended to correlate with the level of radiopollutant in PTC. In contrast to sporadic PTC, highly significant-positive correlation between the presence of large scale mtDNA deletions and relative mtDNA content was found in radiation-associated tumors (P = 0.001 and P = 0.019 in PTC and follicular adenoma, respectively). Normal tissue displayed the inverse tendency. No association with level of the CD was found in either group of cases. Concordant increase of both relative mtDNA content and number of mtDNA deletions was detected more often in radiation-associated PTC than in sporadic PTC. Thus, simultaneous determination of the number of large-scale mtDNA deletions and relative mtDNA content may be useful to elucidate molecular distinctive features of radiation-associated thyroid tumors.

Network analyses based on comprehensive molecular interaction maps reveal robust control structures in yeast stress response pathways.

Cellular stress responses require exquisite coordination between intracellular signaling molecules to integrate multiple stimuli and actuate specific cellular behaviors. Deciphering the web of complex interactions underlying stress responses is a key challenge in understanding robust biological systems and has the potential to lead to the discovery of targeted therapeutics for diseases triggered by dysregulation of stress response pathways. We constructed large-scale molecular interaction maps of six major stress response pathways in Saccharomyces cerevisiae (baker's or budding yeast). Biological findings from over 900 publications were converted into standardized graphical formats and integrated into a common framework. The maps are posted at http://www.yeast-maps.org/yeast-stress-response/ for browse and curation by the research community. On the basis of these maps, we undertook systematic analyses to unravel the underlying architecture of the networks. A series of network analyses revealed that yeast stress response pathways are organized in bow-tie structures, which have been proposed as universal sub-systems for robust biological regulation. Furthermore, we demonstrated a potential role for complexes in stabilizing the conserved core molecules of bow-tie structures. Specifically, complex-mediated reversible reactions, identified by network motif analyses, appeared to have an important role in buffering the concentration and activity of these core molecules. We propose complex-mediated reactions as a key mechanism mediating robust regulation of the yeast stress response. Thus, our comprehensive molecular interaction maps provide not only an integrated knowledge base, but also a platform for systematic network analyses to elucidate the underlying architecture in complex biological systems.

Novel tumorigenic rearrangement, Delta rfp/ret, in a papillary thyroid carcinoma from externally irradiated patient.

Molecular analysis of cDNA derived from a papillary thyroid carcinoma (PTC) (follicular variant of papillary thyroid carcinoma on histology) which developed in an externally irradiated patient 4 years after exposure identified a portion of the 5' region, exons 1-3, of the rfp gene juxtaposed upstream of the fragment encoding the tyrosine kinase (TK) domain of the ret gene. The fusion gene, termed Delta rfp/ret, was the result of a balanced chromosomal translocation t(6;10) (p21.3;q11.2) confirmed by interphase FISH painting, with breakpoints occurring in introns 3 and 11 of the rfp and ret genes, respectively. Both Delta rfp/ret and reciprocal ret/rfp chimeric introns had small deletions around breakpoints consistent with presumed misrepair of a radiation-induced double-strand DNA break underlying the rearrangement. No extensive sequence homology was found between the fragments flanking the breakpoints. The fusion protein retained the propensity to form oligomers likely to be mediated by a coiled-coil of the RFP polypeptide as assessed by a yeast two-hybrid system. NIH 3T3 fibroblasts stably transfected with a mammalian expression vector encoding full-length Delta RFP/RET readily gave rise to the tumors in athymic mice suggestive of high transforming potential of the fusion protein. Thus, the Delta rfp/ret rearrangement may be involved in a causative manner in cancerogenesis and provides additional evidence of the role of activated ret oncogene in the development of a subset of papillary thyroid carcinoma.

Robustness analysis of cellular systems using the genetic tug-of-war method.

Robustness is one of the principles of design inherent to biological systems. Cellular robustness can be measured as limits of intracellular parameters such as gene expression levels. We have recently developed an experimental approach coined as genetic Tug-Of-War (gTOW), which we used to perform robustness analysis in yeast. Using gTOW, we were able to measure the upper limit of expression of gene targets. In this review, we first elaborate on how the gTOW method compares to current mathematical simulation models prevalently used in the determination of robustness. We then explain the experimental principles underlying gTOW and its associated tools, and we provide concrete examples of robustness analysis using gTOW, i.e. cell cycle and HOG pathway gene expression analysis. Finally, we list a series of Q&As related to the experimental utilization of gTOW and we describe the potential impact of gTOW and its relevance to the understanding of biological systems.

Increased expression of nuclear envelope gp210 antigen in small bile ducts in primary biliary cirrhosis.

The sustained antibody response to nuclear envelope gp210 antigen indicates a group of primary biliary cirrhosis (PBC) patients at high risk for the progression to end-stage hepatic failure. To address this issue, we immunohistochemically studied the expression of gp210 antigen in needle liver biopsy specimens from PBC patients using a monoclonal antibody specific for gp210 antigen. The specimens from autoimmune hepatitis (AIH), chronic viral hepatitis B (CHB) and C (CHC) patients served as disease controls. The expression of gp210 antigen was apparently increased on the nuclear envelope of biliary epithelial cells (BECs) of small bile ducts in almost all specimens from PBC. In contrast, the expression of gp210 antigen was negative in BECs of small bile ducts in normal liver, while relatively weak anti-gp210 immunostaining was observed in AIH, CHC and CHB. In addition, the degree of gp210 expression in BECs of small bile ducts was positively correlated to that of portal inflammation, interface hepatitis and lobular inflammation in PBC. These results indicate that the increased expression of gp210 in small bile ducts, which is probably associated with damage to BECs by inflammation, is possibly involved in autoimmune response to gp210 leading to the progression to end-stage hepatic failure in PBC.

Antibody titer to gp210-C terminal peptide as a clinical parameter for monitoring primary biliary cirrhosis.

BACKGROUND/AIMS: The presence of antibodies to the 210-kDa glycoprotein of the nuclear pore complex (gp210) is highly indicative of primary biliary cirrhosis (PBC). However, the significance of anti-gp210 antibody titers for monitoring PBC remains unresolved. METHODS: We used an ELISA with a gp210 C-terminal peptide as an antigen to assess serum antibody titers in 71 patients with PBC. RESULTS: Patients were classified into three groups: Group A in whom anti-gp210 titers were sustained at a high level, Group B in whom anti-gp210 status changed from positive to negative under ursodeoxycholic acid (UDCA) therapy, Group C in whom anti-gp210 antibodies were negative at the time of diagnosis. The rate of progression to end-stage hepatic failure was significantly higher in group A (60%) as compared to groups B (0%) and C (4.2%). The sustained antibody response to gp210 was closely associated with the severity of interface hepatitis. The significance of anti-gp210 antibody was confirmed by National Hospital Organization Study Group for Liver Disease in Japan. CONCLUSIONS: The serial quantitation of serum anti-gp210-C-terminal peptide antibodies is useful for monitoring the effect of UDCA and for the early identification of patients at high risk for end-stage hepatic failure.

Enhanced expression of type I interferon and toll-like receptor-3 in primary biliary cirrhosis.

The pathogenesis of primary biliary cirrhosis (PBC) remains enigmatic. In order to address this issue, we analyzed by laser capture microdissection and real-time reverse transcription-polymerase chain reaction the site-specific expression of messenger RNA (mRNA) for cytokines (interferon (IFN)-alpha, -beta, -gamma, interleukin (IL)-1beta, -4, -6, -10, -12p40, -18, tumor necrosis factor-alpha) and toll-like receptors (TLRs) (TLR-2, -3, -4, -7, -9) in portal tract and liver parenchyma from patients with early-stage PBC. Expression of IFN-alpha, -beta and TLR-3 proteins was also studied by immunohistochemistry. Autoimmune hepatitis (AIH) and chronic hepatitis C (CHC) served as disease controls. The expression levels of type I IFN (IFN-alpha, -beta) and TLR-3 mRNAs, which are known to induce type I IFN, were significantly higher in portal tract and liver parenchyma as compared to AIH and CHC. A strong positive correlation between the mRNA levels of type I IFN and TLR-3 was also seen in both areas. Immunohistologically, IFN-alpha is present in the mononuclear cells in portal tract and sinusoidal cells. Macrophages in portal tract and hepatocytes expressed IFN-beta and TLR-3. Furthermore, the level of IFN-alpha mRNA in the portal tract was positively correlated with serum alkaline phosphatase. In conclusion, these data indicate that TLR-3 and type I IFN signaling pathways are active in both the portal tract and liver parenchyma of early-stage PBC, and form the basis for our hypothesis that these signaling pathways are involved in the pathophysiology of PBC.

Cyclin D1 overexpression in thyroid tumours from a radio-contaminated area and its correlation with Pin1 and aberrant beta-catenin expression.

Cyclin D1 is a target molecule transcriptionally activated by aberrant beta-catenin in Wnt signalling, while prolyl isomerase Pin1 promotes cyclin D1 overexpression directly or through accumulation of beta-catenin in cancer cells. This study aimed to elucidate whether Pin1 was involved in cyclin D1 overexpression and aberrant beta-catenin in thyroid tumourigenesis by examining 14 follicular adenomas (FAa) and 14 papillary thyroid carcinomas (PTCs). All PTCs displayed cyclin D1 overexpression and strong cytoplasmic beta-catenin and/or decreased membrane beta-catenin expression by immunohistochemistry. Overexpression of cyclin D1 mRNA was observed in 45.5% of FAs and 54.5% of PTCs by TaqMan real-time PCR. Pin1 expression was observed in PTC by immunostaining and was confirmed by reverse transcriptase-PCR. There was a strong correlation between cyclin D1 and Pin1/cytoplasmic/membrane beta-catenin expression (p < 0.001), and between Pin1 and cytoplasmic (p < 0.001)/membrane (p = 0.002) beta-catenin expression in thyroid tumours. Mutation of the beta-catenin gene could not be detected in PTC. Western blot analysis demonstrated high levels of cyclin D1 and beta-catenin as well as Pin1 expression in a human PTC cell line possessing wild-type beta-catenin and APC genes. This study suggests that both cyclin D1 overexpression and aberrant beta-catenin expression are of significance in thyroid tumours. Pin1 expression appears to correlate closely with the level of cyclin D1 and aberrant beta-catenin expression in thyroid tumours such as FA and PTC. Pin1 may be an important factor in regulating cyclin D1 and beta-catenin expression during thyroid carcinogenesis.