Methods for measuring rates of protein binding to insoluble scaffolds in living cells: histone H1-chromatin interactions

Understanding of cell regulation is limited by our inability to measure molecular binding rates for proteins within the structural context of living cells, and many systems biology models are hindered because they use values obtained with molecules binding in solution. Here, we present a kinetic analysis of GFP-histone H1 binding to chromatin within nuclei of living cells that allows both the binding rate constant k(ON) and dissociation rate constant k(OFF) to be determined based on data obtained from fluorescence recovery after photobleaching (FRAP) analysis. This is accomplished by measuring the ratio of bound to free concentration of protein at steady state, and identifying the rate-determining step during FRAP recovery experimentally, combined with mathematical modeling. We report k(OFF) = 0.0131/s and k(ON) = 0.14/s for histone H1.1 binding to chromatin. This work brings clarity to the interpretation of FRAP experiments and provides a way to determine binding kinetics for nuclear proteins and other cellular molecules that interact with insoluble scaffolds within living cells.     

One RNA plays three roles to provide catalytic activity to a group I intron lacking an endogenous internal guide sequence

Catalytic RNA molecules possess simultaneously a genotype and a phenotype. However, a single RNA genotype has the potential to adopt two or perhaps more distinct phenotypes as a result of differential folding and/or catalytic activity. Such multifunctionality would be particularly significant if the phenotypes were functionally inter-related in a common biochemical pathway. Here, this phenomenon is demonstrated by the ability of the Azoarcus group I ribozyme to function when its canonical internal guide sequence (GUG) has been removed from the 5′ end of the molecule, and added back exogenously in trans. The presence of GUG triplets in non-covalent fragments of the ribozyme allow trans-splicing to occur in both a reverse splicing assay and a covalent self-assembly assay in which the internal guide sequence (IGS)-less ribozyme can put itself together from two of its component pieces. Analysis of these reactions indicates that a single RNA fragment can perform up to three distinct roles in a reaction: behaving as a portion of a catalyst, behaving as a substrate, and providing an exogenous IGS. This property of RNA to be multifunctional in a single reaction pathway bolsters the probability that a system of self-replicating molecules could have existed in an RNA world during the origins of life on the Earth.     

Inhibition of terminal differentiation of B cells mediated by CD27 and CD40 involves signaling through JNK

B cells responding to cognate Ag in vivo undergo clonal expansion that is followed by differentiation into Ab-secreting plasma cells or into quiescent restimulable memory. Both these events occur in the germinal center and require that cells exit from proliferation, but the signals that lead to one or the other of these mutually exclusive differentiation pathways have not been definitively characterized. Previous experiments have shown that signals transduced through the TNFRs CD27 and CD40 at the time of B cell stimulation in vitro or in vivo can influence this cell fate decision by inhibiting terminal differentiation and promoting memory. In this study, we show that the PIQED domain of the cytoplasmic tail of murine CD27 and the adapter molecule TNFR-associated factor 2 are involved in this effect. Using pharmacological inhibitors of signaling intermediates, we identify JNK as being necessary and sufficient for the observed inhibition of terminal differentiation. While JNK is involved downstream of CD40, inhibition of the MEK pathway can also partially restore plasma cell generation, indicating that both signaling intermediates may be involved. We also show that inhibition of induction of IFN regulatory factor 4 and B lymphocyte induced maturation protein 1 are downstream events common to both receptors.     

Rab11 regulates JNK and Raf/MAPK‐ERK signalling pathways during Drosophila wing development

Developmental signalling pathways are regulated by intracellular vesicle trafficking in multicellular organisms. In our earlier communication, we have shown that mutation in Rab11 (a subfamily of the Ypt/Rab gene family) results in the activation of JNK signalling pathways in Drosophila eye. Here, we report that Rab11 regulates JNK and Raf/MAPK‐ERK signalling pathways during Drosophila wing development. Using immunofluorescence and immunohistochemical analyses, we show that overexpression of Rab11 in mutant wing imaginal disc cells triggers the induction of apoptosis and activation of JNK and ERK. Further, using a genetic approach it has been shown that Rab11 interacts with the components of these pathways during Drosophila wing development. In addition to this, in Rab11 mutant wing imaginal discs JNK activity was monitored using puc^E69, a P‐lacZ enhancer‐trap line inserted in puckered (puc). A strong induction of puc in Rab11 mutant wing imaginal disc cells provided a strong support that Rab11 regulates the JNK signalling pathway during Drosophila wing development.     

Identification of Host-Dependent Survival Factors for Intracellular Mycobacterium tuberculosis through an siRNA Screen

The stable infection of host macrophages by Mycobacterium tuberculosis (Mtb) involves, and depends on, the attenuation of the diverse microbicidal responses mounted by the host cell. This is primarily achieved through targeted perturbations of the host cellular signaling machinery. Therefore, in view of the dependency of the pathogen on host molecules for its intracellular survival, we wanted to test whether targeting such factors could provide an alternate route for the therapeutic management of tuberculosis. To first identify components of the host signaling machinery that regulate intracellular survival of Mtb, we performed an siRNA screen against all known kinases and phosphatases in murine macrophages infected with the virulent strain, H37Rv. Several validated targets could be identified by this method where silencing led either to a significant decrease, or enhancement in the intracellular mycobacterial load. To further resolve the functional relevance of these targets, we also screened against these identified targets in cells infected with different strains of multiple drug-resistant mycobacteria which differed in terms of their intracellular growth properties. The results obtained subsequently allowed us to filter the core set of host regulatory molecules that functioned independently of the phenotypic variations exhibited by the pathogen. Then, using a combination of both in vitro and in vivo experimentation, we could demonstrate that at least some of these host factors provide attractive targets for anti-TB drug development. These results provide a “proof-of-concept” demonstration that targeting host factors subverted by intracellular Mtb provides an attractive and feasible strategy for the development of anti-tuberculosis drugs. Importantly, our findings also emphasize the advantage of such an approach by establishing its equal applicability to infections with Mtb strains exhibiting a range of phenotypic diversifications, including multiple drug-resistance. Thus the host factors identified here may potentially be exploited for the development of anti-tuberculosis drugs.     

In vitro activity of 11 antibiotics against 74 anaerobes isolated from pediatric intra-abdominal infections

The in vitro activity of 11 antimicrobials was tested against 74 recent anaerobic isolates obtained from pretreatment cultures in pediatric patients with complicated intra-abdominal infections using the CLSI M11-A-6 agar dilution method. Carbapenems, beta-lactamase inhibitor combinations and metronidazole retained good activity, while all Bacteroides fragilis group species produced beta-lactamase and were penicillin resistant and 43% were either intermediately susceptible or resistant to clindamycin. Cefoxitin had moderate activity against B. fragilis but poor activity against Bacteroides thetaiotaomicron and other B. fragilis group isolates.     

Agrobacterium is not alone: gene transfer to plants by viruses and other bacteria

Agrobacterium-mediated genetic transformation is the most widely used technology for obtaining the overexpression of recombinant proteins in plants. However, complex patent issues related to the use of Agrobacterium as a tool for plant genetic engineering and the general requirement of establishing transgenic plants can create obstacles in using this technology for speedy research and development and for agricultural improvements in many plant species. Recent studies addressing these issues have shown that virus-based vectors can be efficiently used for high transient expression of foreign proteins in transfected plants and that non-Agrobacterium bacterial species can be used for the production of transgenic plants, laying the foundation for alternative tools for future plant biotechnology.     

Extractive cultivation of recombinant Escherichia coli using aqueous two phase systems for production and separation of extracellular xylanase

Recombinant Escherichia coli (pATBX 1.8) secreting extracellular xylanase was used as a model system to study the application of an aqueous two phase system for extractive cultivation. An increase in the polymer concentrations from 6 to 20% in the polyethylene glycol phosphate aqueous two phase system resulted in an increase in the phase volume ratio with a concomitant decrease in the partition coefficient (K) and recovery of xylanase in the top phase. However, varying phosphate concentrations from 8 to 16% decreased both the phase volume ratio and the partition coefficient of xylanase. The polyethylene glycol (6%) and phosphate (12%) system was found to be optimum for extracellular cultivation of E. coli, where extracellular xylanase was selectively partitioned to the top phase giving a purification ratio of above 1.0. The process was extended to a semicontinuous operating mode at the optimal condition, wherein the top phase containing xylanase was recovered and the surviving cells were recycled together with the new top phase. The maximum recovery of xylanase was obtained after 12 h in the top phase with a twofold increase in the specific activity as compared to the one obtained in the reference fermentation. In the present work, we report for the first time the use of the two phase system for the extractive cultivation of recombinant E. coli (pATBX 1.8) with the purpose of obtaining a simple and inexpensive separation procedure and achieving the maximal extraction of xylanase to one phase.     

The novel method for isolating chitinolytic bacteria and its application in screening for hyperchitinase producing mutant of Alcaligenes xylosoxydans

AIMS: To develop a novel, rapid and effective screening method for chitinase producing bacteria. METHODS AND RESULTS: A simple and rapid technique for screening of potential chitinolytic bacteria has been developed using the chitin binding dye calcofluor white M2R in chitin agar. Microorganisms possessing high chitinolytic potential gave a clear zone under ultraviolet light after 24-48 h of incubation. This method was successfully applied for isolating the hyperchitinase mutant of Alcaligenes xylosoxydans. The mutant Alc. xylosoxydans EMS 33 was found to produce 3.4 times more chitinase than the wild type. CONCLUSIONS: In this study, the screening method for chitinase producing bacteria has been developed and it was applied to screen chitinase-overproducing mutant of Alc. xylosoxydans. SIGNIFICANCE AND IMPACT OF THE STUDY: The novel screening method for chitinase producer is more sensitive, rapid, user-friendly and reliable, which can also be used for screening of recombinants having chitinase gene.