Effect of immobilization support, water activity, and enzyme ionization state on cutinase activity and enantioselectivity in organic media

We studied the reaction between vinyl butyrate and 2-phenyl-1-propanol in acetonitrile catalyzed by Fusarium solani pisi cutinase immobilized on zeolites NaA and NaY and on Accurel PA-6. The choice of 2-phenyl-1-propanol was based on modeling studies that suggested moderate cutinase enantioselectivity towards this substrate. With all the supports, initial rates of transesterification were higher at a water activity (a(w)) of 0.2 than at a(w) = 0.7, and the reverse was true for initial rates of hydrolysis. By providing acid-base control in the medium through the use of solid-state buffers that control the parameter pH-pNa, which we monitored using an organo-soluble chromoionophoric indicator, we were able, in some cases, to completely eliminate dissolved butyric acid. However, none of the buffers used were able to improve the rates of transesterification relative to the blanks (no added buffer) when the enzyme was immobilized at an optimum pH of 8.5. When the enzyme was immobilized at pH 5 and exhibited only marginal activity, however, even a relatively acidic buffer with a pK(a) of 4.3 was able to restore catalytic activity to about 20% of that displayed for a pH of immobilization of 8.5, at otherwise identical conditions. As a(w) was increased from 0.2 to 0.7, rates of transesterification first increased slightly and then decreased. Rates of hydrolysis showed a steady increase in that a(w) range, and so did total initial reaction rates. The presence or absence of the buffers did not impact on the competition between transesterification and hydrolysis, regardless of whether the butyric acid formed remained as such in the reaction medium or was eliminated from the microenvironment of the enzyme through conversion into an insoluble salt. Cutinase enantioselectivity towards 2-phenyl-1-propanol was indeed low and was not affected by differences in immobilization support, enzyme protonation state, or a(w).     

Thinking about Bacillus subtilis as a multicellular organism

Initial attempts to use colony morphogenesis as a tool to investigate bacterial multicellularity were limited by the fact that laboratory strains often have lost many of their developmental properties. Recent advances in elucidating the molecular mechanisms underlying colony morphogenesis have been made possible through the use of undomesticated strains. In particular, Bacillus subtilis has proven to be a remarkable model system to study colony morphogenesis because of its well-characterized developmental features. Genetic screens that analyze mutants defective in colony morphology have led to the discovery of an intricate regulatory network that controls the production of an extracellular matrix. This matrix is essential for the development of complex colony architecture characterized by aerial projections that serve as preferential sites for sporulation. While much progress has been made, the challenge for future studies will be to determine the underlying mechanisms that regulate development such that differentiation occurs in a spatially and temporally organized manner.     

Reduced gain of excitation-contraction coupling in triadin-null myotubes is mediated by the disruption of FKBP12/RyR1 interaction

Several studies have suggested that triadin (Tdn) may be a critical component of skeletal EC-coupling. However, using Tdn-null mice we have shown that triadin ablation results in no significant disruption of skeletal EC-coupling. To analyze the role of triadin in EC-coupling signaling here we used whole-cell voltage clamp and simultaneous recording of intracellular Ca2+ release to characterize the retrograde and orthograde signaling between RyR1 and DHPR in cultured myotubes. DHPR Ca2+ currents elicited by depolarization of Wt and Tdn-null myotubes displayed similar current densities and voltage dependence. However, kinetic analysis of the Ca2+ current shows that activation time constant of the slow component was slightly decreased in Tdn-null cells. Voltage-evoked Ca2+ transient of Tdn-null myotubes showed small but significant reduction in peak fluorescence amplitude but no differences in voltage dependence. This difference in Ca2+ amplitude was averted by over-expression of FKBP12.6. Our results show that bi-directional signaling between DHPR and RyR1 is preserved nearly intact in Tdn-null myotubes and that the effect of triadin ablation on Ca2+ transients appears to be secondary to the reduced FKBP12 binding capacity of RyR1 in Tdn-null myotubes. These data suggest that skeletal triadins do not play a direct role in skeletal EC-coupling.     

Correlation analysis reveals the emergence of coherence in the gene expression dynamics following system perturbation

Time course gene expression experiments are a popular means to infer co-expression. Many methods have been proposed to cluster genes or to build networks based on similarity measures of their expression dynamics. In this paper we apply a correlation based approach to network reconstruction to three datasets of time series gene expression following system perturbation: 1) Conditional, Tamoxifen dependent, activation of the cMyc proto-oncogene in rat fibroblast; 2) Genomic response to nutrition changes in D. melanogaster; 3) Patterns of gene activity as a consequence of ageing occurring over a life-span time series (25y-90y) sampled from T-cells of human donors. We show that the three datasets undergo similar transitions from an "uncorrelated" regime to a positively or negatively correlated one that is symptomatic of a shift from a "ground" or "basal" state to a "polarized" state. In addition, we show that a similar transition is conserved at the pathway level, and that this information can be used for the construction of "meta-networks" where it is possible to assess new relations among functionally distant sets of molecular functions.     

Seasonal fluctuations in Vitis vinifera root respiration in the field

We studied the seasonal fluctuation of soil respiration (R(S)), and its root-dependent (R(R)) and basal (R(B)) components, in a Vitis vinifera (Chardonnay) vineyard. The R(S) components were estimated through independent field methods (y-intercept and trenching) and modeled on the basis of a Q(10) response to soil temperature, and fine and coarse root respiration coefficients. The effect of assimilate availability on R(R) was assessed through a trunk girdling treatment. The apparent Q(10) for R(R) was twice that of R(B) (3.5 vs 1.6) and increased linearly with increasing vine root biomass. The fastest R(R) of fine roots was during rapid fruit growth and the fastest R(R) of coarse roots was immediately following fruit development. R(S) was estimated at 32.6 kg ha(-1) d(-1) (69% as a result of R(R) ) for the hottest month and at 7.6 kg ha(-1) d(-1) (18% as a result of R(R)) during winter dormancy. Annual R(S) was low compared with other natural and cultivated ecosystems: 5.4 Mg ha(-1) (46% as a result of R(R)). Our estimates of annual vineyard R(S) are the first for any horticultural crop and suggest that the assumption that they are similar to those of annual crops or forest trees might lead to an overestimation.     

Connective tissue growth factor induction by lysophosphatidic acid requires transactivation of transforming growth factor type β receptors and the JNK pathway

Transforming growth factor β (TGF-β) is a very strong pro-fibrotic factor which mediates its action, at least in part, through the expression of connective tissue growth factor (CTGF/CCN2). Along with these cytokines, the involvement of phospholipids in wound healing and the development of fibrosis has been revealed. Among them, lysophosphatidic acid (LPA) is a novel, potent regulator of wound healing and fibrosis that has diverse effects on many types of cells. We decided to evaluate the effect of LPA together with TGF-β on CTGF expression. We found that myoblasts treated with LPA and TGF-β1 produced an additive effect on CTGF expression. In the absence of TGF-β, the induction of CTGF expression by LPA was abolished by a dominant negative form of the TGF-β receptor type II (TGF-βRII) and by the use of SB 431542, a specific inhibitor of the serine/threonine kinase activity of TGF-βRI, suggesting that CTGF induction is dependent on LPA and requires active TGF-βRs. Moreover, we show that LPA requires Smad-2/3 proteins for the induction of CTGF expression, but not their phosphorylation or their nuclear translocation. The requirement of TGF-βRI for LPA mediated-effects is differential, since treatment of myoblasts with LPA in the presence of SB 431542 abolished the induction of stress fibers but not the induction of proliferation. Finally, we demonstrated that CTGF induction in response to LPA requires the activation of JNK, but not ERK, signaling pathways. The JNK requirement is independent of TGF-βRI-mediated activity. These novel results for the mechanism of action of LPA and TGF-β are important for understanding the role of pro-fibrotic growth factors and phospholipids involved in wound healing and related diseases.     

Abnormal kinetochore-generated pulling forces from expressing a N-terminally modified Hec1

Background

Highly Expressed in Cancer protein 1 (Hec1) is a constituent of the Ndc80 complex, a kinetochore component that has been shown to have a fundamental role in stable kinetochore-microtubule attachment, chromosome alignment and spindle checkpoint activation at mitosis. HEC1 RNA is found up-regulated in several cancer cells, suggesting a role for HEC1 deregulation in cancer. In light of this, we have investigated the consequences of experimentally-driven Hec1 expression on mitosis and chromosome segregation in an inducible expression system from human cells.

Methodology/Principal Findings

Overexpression of Hec1 could never be obtained in HeLa clones inducibly expressing C-terminally tagged Hec1 or untagged Hec1, suggesting that Hec1 cellular levels are tightly controlled. On the contrary, a chimeric protein with an EGFP tag fused to the Hec1 N-terminus accumulated in cells and disrupted mitotic division. EGFP- Hec1 cells underwent altered chromosome segregation within multipolar spindles that originated from centriole splitting. We found that EGFP-Hec1 assembled a mutant Ndc80 complex that was unable to rescue the mitotic phenotypes of Hec1 depletion. Kinetochores harboring EGFP-Hec1 formed persisting lateral microtubule-kinetochore interactions that recruited the plus-end depolymerase MCAK and the microtubule stabilizing protein HURP on K-fibers. In these conditions the plus-end kinesin CENP-E was preferentially retained at kinetochores. RNAi-mediated CENP-E depletion further demonstrated that CENP-E function was required for multipolar spindle formation in EGFP-Hec1 expressing cells.

Conclusions/Significance

Our study suggests that modifications on Hec1 N-terminal tail can alter kinetochore-microtubule attachment stability and influence Ndc80 complex function independently from the intracellular levels of the protein. N-terminally modified Hec1 promotes spindle pole fragmentation by CENP-E-mediated plus-end directed kinetochore pulling forces that disrupt the fine balance of kinetochore- and centrosome-associated forces regulating spindle bipolarity. Overall, our findings support a model in which centrosome integrity is influenced by the pathways regulating kinetochore-microtubule attachment stability.     

Characterization and functional activity of murine monoclonal antibodies specific for α1,6-glucan chain of Helicobacter pylori lipopolysaccharide

Background: The outer core region of H. pylori lipopolysaccharide (LPS) contains α1,6‐glucan previously shown to contribute to colonizing efficiency of a mouse stomach. The aim of the present study was to generate monoclonal antibodies (mAbs) specific for α1,6‐glucan and characterize their binding properties and functional activity. Materials and Methods: BALB/c mice were injected intraperitoneally with 10⁸ formalin‐fixed H. pylori O:3 0826::Kan cells 3× over 56 days to achieve significant titer. Anti‐α1,6‐glucan‐producing hybridomas were screened by indirect ELISA using purified H. pylori O:3 0826::Kan LPS. One clone, 1C4F9, was selected for further characterization. The specificities of mAbs were determined by indirect and inhibition ELISA using structurally defined H. pylori LPS and synthetic oligosaccharides, and whole‐cell indirect ELISA (WCE) of clinical isolates. They were further characterized by indirect immunofluorescent (IF) microscopy and their functional activity in vitro determined by serum bactericidal assays against wild‐type and mutant strains of H. pylori. Results: The generated anti‐α1,6‐glucan IgM, 1C4F9, has demonstrated an excellent specificity for the glucan chain containing 5 to 6 α1,6‐linked glucose residues and showed surface accessibility by IF microscopy with H. pylori cells adherent to gastric adenocarcinoma cells monolayers. Of 38 isolates from Chile, 17 strains reacted with antiglucan mAbs in WCE (OD₄₅₀ ≥ 0.2). Bactericidal activity was observed against selective wild‐type and mutant H. pylori strains exhibiting OD₄₅₀ values of ≥0.45 in WCE. Conclusions: Anti‐α1,6‐glucan mAbs could have potential application in typing and surveillance of H. pylori isolates as well as offer insights into structural requirements for the development of LPS‐based vaccine against H. pylori infections.