The Goldbeter-Koshland switch in the first-order region and its response to dynamic disorder

In their classical work (Proc. Natl. Acad. Sci. USA, 1981, 78:6840-6844), Goldbeter and Koshland mathematically analyzed a reversible covalent modification system which is highly sensitive to the concentration of effectors. Its signal-response curve appears sigmoidal, constituting a biochemical switch. However, the switch behavior only emerges in the "zero-order region", i.e. when the signal molecule concentration is much lower than that of the substrate it modifies. In this work we showed that the switching behavior can also occur under comparable concentrations of signals and substrates, provided that the signal molecules catalyze the modification reaction in cooperation. We also studied the effect of dynamic disorders on the proposed biochemical switch, in which the enzymatic reaction rates, instead of constant, appear as stochastic functions of time. We showed that the system is robust to dynamic disorder at bulk concentration. But if the dynamic disorder is quasi-static, large fluctuations of the switch response behavior may be observed at low concentrations. Such fluctuation is relevant to many biological functions. It can be reduced by either increasing the conformation interconversion rate of the protein, or correlating the enzymatic reaction rates in the network.Comment: 23 pages, 4 figures, accepted by PLOS ON

Proteomic profile of KSR1-regulated signalling in response to genotoxic agents in breast cancer

Kinase suppressor of Ras 1 (KSR1) has been implicated in tumorigenesis in multiple cancers, including skin, pancreatic and lung carcinomas. However, our recent study revealed a role of KSR1 as a tumour suppressor in breast cancer, the expression of which is potentially correlated with chemotherapy response. Here, we aimed to further elucidate the KSR1-regulated signalling in response to genotoxic agents in breast cancer. Stable isotope labelling by amino acids in cell culture (SILAC) coupled to high-resolution mass spectrometry (MS) was implemented to globally characterise cellular protein levels induced by KSR1 in the presence of doxorubicin or etoposide. The acquired proteomic signature was compared and GO-STRING analysis was subsequently performed to illustrate the activated functional signalling networks. Furthermore, the clinical associations of KSR1 with identified targets and their relevance in chemotherapy response were examined in breast cancer patients. We reveal a comprehensive repertoire of thousands of proteins identified in each dataset and compare the unique proteomic profiles as well as functional connections modulated by KSR1 after doxorubicin (Doxo-KSR1) or etoposide (Etop-KSR1) stimulus. From the up-regulated top hits, several proteins, including STAT1, ISG15 and TAP1 are also found to be positively associated with KSR1 expression in patient samples. Moreover, high KSR1 expression, as well as high abundance of these proteins, is correlated with better survival in breast cancer patients who underwent chemotherapy. In aggregate, our data exemplify a broad functional network conferred by KSR1 with genotoxic agents and highlight its implication in predicting chemotherapy response in breast cancer

SILAC-based phosphoproteomics reveals an inhibitory role of KSR1 in p53 transcriptional activity via modulation of DBC1

BACKGROUND We have previously identified kinase suppressor of ras-1 (KSR1) as a potential regulatory gene in breast cancer. KSR1, originally described as a novel protein kinase, has a role in activation of mitogen-activated protein kinases. Emerging evidence has shown that KSR1 may have dual functions as an active kinase as well as a scaffold facilitating multiprotein complex assembly. Although efforts have been made to study the role of KSR1 in certain tumour types, its involvement in breast cancer remains unknown. METHODS A quantitative mass spectrometry analysis using stable isotope labelling of amino acids in cell culture (SILAC) was implemented to identify KSR1-regulated phosphoproteins in breast cancer. In vitro luciferase assays, co-immunoprecipitation as well as western blotting experiments were performed to further study the function of KSR1 in breast cancer. RESULTS Of significance, proteomic analysis reveals that KSR1 overexpression decreases deleted in breast cancer-1 (DBC1) phosphorylation. Furthermore, we show that KSR1 decreases the transcriptional activity of p53 by reducing the phosphorylation of DBC1, which leads to a reduced interaction of DBC1 with sirtuin-1 (SIRT1); this in turn enables SIRT1 to deacetylate p53. CONCLUSION Our findings integrate KSR1 into a network involving DBC1 and SIRT1, which results in the regulation of p53 acetylation and its transcriptional activity

Ratio-Based Analysis of Differential mRNA Processing and Expression of a Polyadenylation Factor Mutant pcfs4 Using Arabidopsis Tiling Microarray

US National Institutes of Health [1R15GM07719201A1]; US National Science Foundation [IOS-0817818]; Ohio Plant Biotech Consortium; National Natural Science Foundation of China [60774033]; Specialized Research Fund for the Doctoral Program of Higher EducatiBackground: Alternative polyadenylation as a mechanism in gene expression regulation has been widely recognized in recent years. Arabidopsis polyadenylation factor PCFS4 was shown to function in leaf development and in flowering time control. The function of PCFS4 in controlling flowering time was correlated with the alternative polyadenylation of FCA, a flowering time regulator. However, genetic evidence suggested additional targets of PCFS4 that may mediate its function in both flowering time and leaf development. Methodology/Principal Findings: To identify further targets, we investigated the whole transcriptome of a PCFS4 mutant using Affymetrix Arabidopsis genomic tiling 1.0R array and developed a data analysis pipeline, termed RADPRE (Ratio-based Analysis of Differential mRNA Processing and Expression). In RADPRE, ratios of normalized probe intensities between wild type Columbia and a pcfs4 mutant were first generated. By doing so, one of the major problems of tiling array data-variations caused by differential probe affinity-was significantly alleviated. With the probe ratios as inputs, a hierarchy of statistical tests was carried out to identify differentially processed genes (DPG) and differentially expressed genes (DEG). The false discovery rate (FDR) of this analysis was estimated by using the balanced random combinations of Col/pcfs4 and pcfs4/Col ratios as inputs. Gene Ontology (GO) analysis of the DPGs and DEGs revealed potential new roles of PCFS4 in stress responses besides flowering time regulation. Conclusion/Significance: We identified 68 DPGs and 114 DEGs with FDR at 1% and 2%, respectively. Most of the 68 DPGs were subjected to alternative polyadenylation, splicing or transcription initiation. Quantitative PCR analysis of a set of DPGs confirmed that most of these genes were truly differentially processed in pcfs4 mutant plants. The enriched GO term "regulation of flower development'' among PCFS4 targets further indicated the efficacy of the RADPRE pipeline. This simple but effective program is available upon request

Analysis of pharmaceuticals in wastewater and removal using a membrane bioreactor

Much attention has recently been devoted to the life and behaviour of pharmaceuticals in the water cycle. In this study the behaviour of several pharmaceutical products in different therapeutic categories (analgesics and anti-inflammatory drugs, lipid regulators, antibiotics, etc.) was monitored during treatment of wastewater in a laboratory-scale membrane bioreactor (MBR). The results were compared with removal in a conventional activated-sludge (CAS) process in a wastewater-treatment facility. The performance of an MBR was monitored for approximately two months to investigate the long-term operational stability of the system and possible effects of solids retention time on the efficiency of removal of target compounds. Pharmaceuticals were, in general, removed to a greater extent by the MBR integrated system than during the CAS process. For most of the compounds investigated the performance of MBR treatment was better (removal rates >80%) and effluent concentrations of, e.g., diclofenac, ketoprofen, ranitidine, gemfibrozil, bezafibrate, pravastatin, and ofloxacin were steadier than for the conventional system. Occasionally removal efficiency was very similar, and high, for both treatments (e.g. for ibuprofen, naproxen, acetaminophen, paroxetine, and hydrochlorothiazide). The antiepileptic drug carbamazepine was the most persistent pharmaceutical and it passed through both the MBR and CAS systems untransformed. Because there was no washout of biomass from the reactor, high-quality effluent in terms of chemical oxygen demand (COD), ammonium content (N-NH(4)), total suspended solids (TSS), and total organic carbon (TOC) was obtained

Facilitating functional annotation of chicken microarray data

<p>Abstract</p> <p>Background</p> <p>Modeling results from chicken microarray studies is challenging for researchers due to little functional annotation associated with these arrays. The Affymetrix GenChip chicken genome array, one of the biggest arrays that serve as a key research tool for the study of chicken functional genomics, is among the few arrays that link gene products to Gene Ontology (GO). However the GO annotation data presented by Affymetrix is incomplete, for example, they do not show references linked to manually annotated functions. In addition, there is no tool that facilitates microarray researchers to directly retrieve functional annotations for their datasets from the annotated arrays. This costs researchers amount of time in searching multiple GO databases for functional information.</p> <p>Results</p> <p>We have improved the breadth of functional annotations of the gene products associated with probesets on the Affymetrix chicken genome array by 45% and the quality of annotation by 14%. We have also identified the most significant diseases and disorders, different types of genes, and known drug targets represented on Affymetrix chicken genome array. To facilitate functional annotation of other arrays and microarray experimental datasets we developed an Array GO Mapper (<it>AGOM</it>) tool to help researchers to quickly retrieve corresponding functional information for their dataset.</p> <p>Conclusion</p> <p>Results from this study will directly facilitate annotation of other chicken arrays and microarray experimental datasets. Researchers will be able to quickly model their microarray dataset into more reliable biological functional information by using <it>AGOM </it>tool. The disease, disorders, gene types and drug targets revealed in the study will allow researchers to learn more about how genes function in complex biological systems and may lead to new drug discovery and development of therapies. The GO annotation data generated will be available for public use via AgBase website and will be updated on regular basis.</p

Expression and clinical significance of Glucose Regulated Proteins GRP78 (BiP) and GRP94 (GP96) in human adenocarcinomas of the esophagus

<p>Abstract</p> <p>Background</p> <p>Glucose regulated proteins (GRPs) are main regulators of cellular homeostasis due to their role as molecular chaperones. Moreover, the functions of GRPs suggest that they also may play important roles in cancer biology. In this study we investigated the glucose regulated proteins GRP78 (BiP) and GRP94 (GP96) in a series of human esophageal adenocarcinomas to determine their implications in cancer progression and prognosis.</p> <p>Methods</p> <p>Formalin-fixed, paraffin-embedded tissues of primary resected esophageal (Barrett) adenocarcinomas (n = 137) and corresponding normal tissue were investigated. mRNA-gene expression levels of GRP78 and GRP94 were determined by quantitative real-time RT-PCR after mRNA extraction. Protein expression analysis was performed with immunohistochemical staining of the cases, assembled on a tissue micorarray. The results were correlated with pathologic features (pT, pN, G) and overall survival.</p> <p>Results</p> <p>GRP78 and GRP94 mRNA were expressed in all tumors. The relative gene expression of GRP78 was significantly higher in early cancers (pT1m and pT1sm) as compared to more advanced stages (pT2 and pT3) and normal tissue (p = 0.031). Highly differentiated tumors showed also higher GRP78 mRNA levels compared to moderate and low differentiated tumors (p = 0.035). In addition, patients with higher GRP78 levels tended to show a survival benefit (p = 0.07). GRP94 mRNA-levels showed no association to pathological features or clinical outcome.</p> <p>GRP78 and GRP94 protein expression was detectable by immunohistochemistry in all tumors. There was a significant correlation between a strong GRP78 protein expression and early tumor stages (pT1m and pT1sm, p = 0.038). For GRP94 low to moderate protein expression was significantly associated with earlier tumor stage (p = 0.001) and less lymph node involvement (p = 0.036). Interestingly, the patients with combined strong GRP78 and GRP94 protein expression exclusively showed either early (pT1m or pT1sm) or advanced (pT3) tumor stages and no pT2 stage (p = 0.031).</p> <p>Conclusion</p> <p>We could demonstrate an association of GRP78 and GRP94 mRNA and protein expression with tumor stage and behaviour in esophageal adenocarcinomas. Increased expression of GRP78 may be responsible for controlling local tumor growth in early tumor stages, while high expression of GRP78 and GRP94 in advanced stages may be dependent from other factors like cellular stress reactions due to glucose deprivation, hypoxia or the hosts' immune response.</p

Revisiting the pH-gated conformational switch on the activities of HisKA-family histidine kinases

Histidine is a versatile residue playing key roles in enzyme catalysis thanks to the chemistry of its imidazole group that can serve as nucleophile, general acid or base depending on its protonation state. In bacteria, signal transduction relies on two-component systems (TCS) which comprise a sensor histidine kinase (HK) containing a phosphorylatable catalytic His with phosphotransfer and phosphatase activities over an effector response regulator. Recently, a pH-gated model has been postulated to regulate the phosphatase activity of HisKA HKs based on the pH-dependent rotamer switch of the phosphorylatable His. Here, we have revisited this model from a structural and functional perspective on HK853-RR468 and EnvZ-OmpR TCS, the prototypical HisKA HKs. We have found that the rotamer of His is not influenced by the environmental pH, ruling out a pH-gated model and confirming that the chemistry of the His is responsible for the decrease in the phosphatase activity at acidic pH

Drugs and herbs given to prevent hepatotoxicity of tuberculosis therapy: systematic review of ingredients and evaluation studies

Background: Drugs to protect the liver are frequently prescribed in some countries as part of treatment for tuberculosis. The biological rationale is not clear, they are expensive and may do harm. We conducted a systematic review to a) describe the ingredients of "liver protection drugs"; and b) compare the evidence base for the policy against international standards. Methods: We searched international medical databases (MEDLINE, EMBASE, LILACS, CINAHL, Cochrane Central Register of Controlled Trials, and the specialised register of the Cochrane Infectious Diseases Group) and Chinese language databases (CNKI, VIP and WanFang) to April 2007. Our inclusion criteria were research papers that reported evaluating any liver protection drug or drugs for preventing liver damage in people taking anti-tuberculosis treatment. Two authors independently categorised and extracted data, and appraised the stated methods of evaluating their effectiveness. Results: Eighty five research articles met our inclusion criteria, carried out in China (77), India (2), Russia (4), Ukraine (2). These articles evaluated 30 distinct types of liver protection compounds categorised as herbal preparations, manufactured herbal products, combinations of vitamins and other non-herbal substances and manufactured pharmaceutical preparations. Critical appraisal of these articles showed that all were small, poorly conducted studies, measuring intermediate outcomes. Four trials that were described as randomised controlled trials were small, had short follow up, and did not meet international standards. Conclusion: There is no reliable evidence to support prescription of drugs or herbs to prevent liver damage in people on tuberculosis treatment