On the specificity of interaction between the Saccharomyces cerevisiae clamp loader replication factor C and primed DNA templates during DNA replication

Replication factor C (RFC) catalyzes assembly of circular proliferating cell nuclear antigen clamps around primed DNA, enabling processive synthesis by DNA polymerase during DNA replication and repair. In order to perform this function efficiently, RFC must rapidly recognize primed DNA as the substrate for clamp assembly, particularly during lagging strand synthesis. Earlier reports as well as quantitative DNA binding experiments from this study indicate, however, that RFC interacts with primer-template as well as single- and double-stranded DNA (ssDNA and dsDNA, respectively) with similar high affinity (apparent K(d) approximately 10 nm). How then can RFC distinguish primed DNA sites from excess ssDNA and dsDNA at the replication fork? Further analysis reveals that despite its high affinity for various DNA structures, RFC selects primer-template DNA even in the presence of a 50-fold excess of ssDNA and dsDNA. The interaction between ssDNA or dsDNA and RFC is far less stable than between primed DNA and RFC (k(off) > 0.2 s(-1) versus 0.025 s(-1), respectively). We propose that the ability to rapidly bind and release single- and double-stranded DNA coupled with selective, stable binding to primer-template DNA allows RFC to scan DNA efficiently for primed sites where it can pause to initiate clamp assembly.     

Extracellular matrix as a contextual determinant of transforming growth factor-β signaling in epithelial-mesenchymal transition and in cancer

Extracellular matrix (ECM) provides both structural support and contextual information to cells within tissues and organs. The combination of biochemical and biomechanical signals from the ECM modulates responses to extracellular signals toward differentiation, proliferation, or apoptosis; alterations in the ECM are necessary for development and remodeling processes, but aberrations in the composition and organization of ECM are associated with disease pathology and can predispose to development of cancer. The primary cell surface sensors of the ECM are the integrins, which provide the physical connection between the ECM and the cytoskeleton and also convey biochemical information about the composition of the ECM. Transforming growth factor-β (TGF-β) is an extracellular signaling molecule that is a powerful controller of a variety of cellular functions, and that has been found to induce very different outcomes according to cell type and cellular context. It is becoming clear that ECM-mediated signaling through integrins is reciprocally influenced by TGF-β: integrin expression, activation, and responses are affected by cellular exposure to TGF-β, and TGF-β activation and cellular responses are in turn controlled by signaling from the ECM through integrins. Epithelial-mesenchymal transition (EMT), a physiological process that is activated by TGF-β in normal development and in cancer, is also affected by the composition and structure of the ECM. Here, we will outline how signaling from the ECM controls the contextual response to TGF-β, and how this response is selectively modulated during disease, with an emphasis on recent findings, current challenges, and future opportunities.     

Detection of polyglutamine protein oligomers in cells by fluorescence correlation spectroscopy

Abnormal aggregation of misfolded proteins and their deposition as inclusion bodies in the brain have been implicated as a common molecular pathogenesis of neurodegenerative diseases including Alzheimer, Parkinson, and the polyglutamine (poly(Q)) diseases, which are collectively called the conformational diseases. The poly(Q) diseases, including Huntington disease and various types of spinocerebellar ataxia, are caused by abnormal expansions of the poly(Q) stretch within disease-causing proteins, which triggers the disease-causing proteins to aggregate into insoluble beta-sheet-rich amyloid fibrils. Although oligomeric structures formed in vitro are believed to be more toxic than mature amyloid fibrils in these diseases, the existence of oligomers in vivo has remained controversial. To explore oligomer formation in cells, we employed fluorescence correlation spectroscopy (FCS), which is a highly sensitive technique for investigating the dynamics of fluorescent molecules in solution. Here we demonstrate direct evidence for oligomer formation of poly(Q)-green fluorescent protein (GFP) fusion proteins expressed in cultured cells, by showing a time-dependent increase in their diffusion time and particle size by FCS. We show that the poly(Q)-binding peptide QBP1 inhibits poly(Q)-GFP oligomer formation, whereas Congo red only inhibits the growth of oligomers, but not the initial formation of the poly(Q)-GFP oligomers, suggesting that FCS is capable of identifying poly(Q) oligomer inhibitors. We therefore conclude that FCS is a useful technique to monitor the oligomerization of disease-causing proteins in cells as well as its inhibition in the conformational diseases.     

Directing pluripotent cell differentiation using "diced RNA" in transient transfection

Embryonic stem (ES) and embryonic carcinoma (EC) cells are pluripotent and have the capacity to differentiate into many cell types. The ability to direct their differentiation should have considerable practical applications. Here, we first report the use of diced short interfering RNAi against Oct4 in a transient approach, to direct differentiation of ES towards the trophectoderm lineage. We then apply this approach to downregulate Smad4 in mouse P19 EC cells. We have found that this leads to an increase in the levels of Pax6 (a neuroectoderm marker), reduction in the levels of Brachyury (a mesoderm marker), and a 3-fold increase in the number of betaIII tubulin-positive colonies when these cells were allowed to differentiate. This indicates a redirection of cell fate towards the neuroectoderm lineage. Thus, transient RNAi could provide a valuable tool to direct pluripotent cells along specific pathways of differentiation while circumventing permanent genetic changes.     

‘Click’ chemistry synthesis and capillary electrophoresis study of 1,4‐linked 1,2,3‐triazole AZT‐systemin conjugate

The Cu(I) catalyzed Huisgen 1,3‐dipolar azide‐alkyne cycloaddition (CuAAC) was applied for a nucleoside‐peptide bioconjugation. Systemin (Sys), an 18‐aa plant signaling peptide naturally produced in response to wounding or pathogen attack, was chemically synthesized as its N‐propynoic acid functionalized analog (Prp‐Sys) using the SPPS. Next, CuAAC was applied to conjugate Prp‐Sys with 3′‐azido‐2′,3′‐dideoxythymidine (AZT), a model cargo molecule. 1,4‐Linked 1,2,3‐triazole AZT‐Sys conjugate was designed to characterize the spreading properties and ability to translocate of cargo molecules of systemin. CuAAC allowed the synthesis of the conjugate in a chemoselective and regioselective manner, with high purity and yield. The presence of Cu(I) ions generated in situ drove the CuAAC reaction to completion within a few minutes without any by‐products. Under typical separation conditions of phosphate ‘buffer’ at low pH and uncoated fused bare‐silica capillary, an increasing peak intensity assigned to triazole‐linked AZT‐Sys conjugate was observed using capillary electrophoresis (CE) during CuAAC. CE analysis showed that systemin peptides are stable in tomato leaf extract for up to a few hours. CE‐ESI‐MS revealed that the native Sys and its conjugate with AZT are translocated through the tomato stem and can be directly detected in stem exudates. The results show potential application of systemin as a transporter of low molecular weight cargo molecules in tomato plant and of CE method to characterize a behavior of plant peptides and its analogs. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Chemerin Is an Antimicrobial Agent in Human Epidermis

Chemerin, a chemoattractant ligand for chemokine-like receptor 1 (CMKLR1) is predicted to share similar tertiary structure with antibacterial cathelicidins. Recombinant chemerin has antimicrobial activity. Here we show that endogenous chemerin is abundant in human epidermis, and that inhibition of bacteria growth by exudates from organ cultures of primary human skin keratinocytes is largely chemerin-dependent. Using a panel of overlapping chemerin-derived synthetic peptides, we demonstrate that the antibacterial activity of chemerin is primarily mediated by Val⁶⁶-Pro⁸⁵, which causes direct bacterial lysis. Therefore, chemerin is an antimicrobial agent in human skin.     

Gas chromatographic–mass spectrometric investigation of metabolites from the needles and roots of pine seedlings at early stages of pathogenic fungi <Emphasis Type="Italic">Armillaria ostoyae</Emphasis> attack

An investigation was carried out of the composition of metabolites in pine seedlings tissues at the initial stages of the infectious process caused by pathogenic fungi Armillaria ostoyae, which causes a root rot of trees and degradation of forest resources. With the help of successive extraction with organic solvents of different polarity, more than 190 metabolites were extracted from the needles and roots of the seedlings and then identified by GC–MS method. The composition of the extracts from control plants and those inoculated with Armillaria ostoyae were compared. It was established that part of secondary metabolites (glucosamines and free amino acids, carbohydrates raffinose and trehalose) were present only in the tissues of inoculated plants. Possible roles of some of these compounds appearing in the roots of seedlings infected with the fungus are also discussed in the paper.     

PseudoMLSA: a database for multigenic sequence analysis of <Emphasis Type="Italic">Pseudomonas</Emphasis> species

Background  

The genus Pseudomonas comprises more than 100 species of environmental, clinical, agricultural, and biotechnological interest. Although, the recommended method for discriminating bacterial species is DNA-DNA hybridisation, alternative techniques based on multigenic sequence analysis are becoming a common practice in bacterial species discrimination studies. Since there is not a general criterion for determining which genes are more useful for species resolution; the number of strains and genes analysed is increasing continuously. As a result, sequences of different genes are dispersed throughout several databases. This sequence information needs to be collected in a common database, in order to be useful for future identification-based projects.     

Androgens drive divergent responses to salt stress in male versus female rat kidneys

Dahl-Iwai (DI) salt-sensitive rats were studied using microarrays to identify sex-specific differences in the kidney, both basal differences and differences in responses to a high-salt diet. In DI rat kidneys, gene expression profiles demonstrated inflammatory and fibrotic responses selectively in females. Gonadectomy of DI rats abrogated sex differences in gene expression. Gonadectomized female and gonadectomized male DI rats both responded to high salt with the same spectrum of gene expression changes as intact female DI rats. Androgens dominated the sex-selective responses to salt. Several androgen-responsive genes with roles potentiating the differential responses to salt were identified, including increased male expression of angiotensin-vasopressin receptor and prolactin receptor, decreased 5 alpha-reductase, and mixed increases and decreases in expression of Cyp4a genes that can produce eicosanoid hormones. These sex differences potentiate sodium retention by males and increase kidney function during gestation in females.     

Acceptability of different species of Brassicaceae as hosts for the cabbage aphid

The probing and feeding behaviour of the cabbage aphid, Brevicoryne brassicae (L.), (Homoptera, Aphididae) was studied on several plant species that represented various levels of acceptability: Sinapis alba L. (a permanent host plant), Capsella bursa-pastoris (L.) Med., Thlaspi arvense L., Lunaria annua L., Erysimum cheiranthoides L. (accidental host plants), Vicia faba L. (a non-host plant), using the electrical penetration graph technique (EPG). B. brassicae on V. faba did not show any patterns related to penetration of phloem vessels. Stylet penetration was deterred on L. annua and E. cheiranthoides where non-penetration prevailed, the periods of sap ingestion were short or did not occur, the percentage of time spent in the phloem was consistently low (5–6%) and E1 salivation predominated. The pathway activities were not suppressed on C. bursa-pastoris and T. arvense and the aphids spent an average of 3 h in the phloem during the 8-h experiment. However, a considerable delay between finding and accepting the phloem and a substantial proportion of E1 salivation (20–30% of all phloem activities) indicated a deterrent factor in the sieve elements of these plants. Aphid probing and sap ingestion were rarely interrupted on S. alba. The results of this study suggest that the deterrent agents vary in activity and may hinder stylet penetration at different levels (epidermis, parenchymatous tissues and/or phloem elements), depending on the plant species.