Probing the proteome response to toluene exposure in the solvent tolerant Pseudomonas putida S12

To enhance target production from biocatalysts, it is necessary to thoroughly understand the molecular mechanisms involved in production, degradation, and, importantly, adaptation to the required environment. One such bacterium with high potential for biocatalysis is the solvent-tolerant bacteria Pseudomonas putida S12, which, among others, is able to degrade organic solvents. For bioconversion of organic solvents to become a successful industrial process, the understanding of the molecular response upon solvent tolerance is essential. Here we performed a quantitative analysis of the P. putida S12 proteome at different stages of adaptation to toluene. Using a stable isotope dimethylation labeling approach we monitored the differential expression of 528 proteins, including often hard-to-detect membrane associate proteins, such as multiple RND-family transporters and ABC transporters of nutrients. Our quantitative proteomics approach revealed the remarkable ability of P. putida S12 to severely change its protein expression profile upon toluene exposure. This proteome response entails a significant increase in energy metabolism and expression of the solvent efflux pump SrpABC, confirming its role in solvent tolerance. Other proteins strongly up-regulated in the presence of toluene include the multidrug efflux membrane protein PP1272 and the cation/acetate symporter ActP and may form interesting alternative targets for improving solvent tolerance.     

Do Size and Insecticide Treatment Matter? Evaluation of Different Nets against Phlebotomus argentipes,the Vector of Visceral Leishmaniasis in Nepal

In the Indian subcontinent, Leishmania donovani, the parasite causing visceral leishmaniasis (VL) is transmitted by the sand fly vector Phlebotomus argentipes. Long lasting insecticide treated nets (LN) have been postulated as alternative or complement to Indoor Residual Spraying but there are few field studies evaluating the entomological efficacy of different nets against this vector. We conducted two crossover trials in a VL endemic area in Nepal to compare the barrier effect of (1) LN with different mesh sizes (156 holes/inch² vs 625 holes/inch²) and (2) alpha-cypermethrin treated LN and untreated nets having the same mesh size (156 holes/inch²). Each crossover trial had two arms consisting of a sequence of two different nets for 8 nights. We used 10 cattle sheds per trial. A cow placed under the net was used as bait. CDC light traps placed inside the nets were used to evaluate the number of P. argentipes crossing the net barrier. Negative binomial generalized estimating equation (GEE) population-averaged models adjusted by night and sequence were used to estimate the barrier effect of the different nets. The crossover trials conducted in a rural village in Morang district (South-eastern Nepal) demonstrated that reducing the size of the holes in treated nets (625 holes/inch²) increased the barrier effect of LN by 77% (95% confidence interval (CI): 56%–88%) compared with treated nets with larger holes (156 holes/inch²). Treating nets with alpha-cypermethrin reduced the number of P. argentipes captured inside the nets by 77% (95% CI: 27%–93%) compared with untreated nets. The effectiveness and acceptability of finer mesh pyrethroid treated LN should be tested for VL prevention in a randomized controlled trial.     

Versican G3 promotes mouse mammary tumor cell growth, migration, and metastasis by influencing EGF receptor signaling

Increased versican expression in breast tumors is predictive of relapse and has negative impact on survival rates. The C-terminal G3 domain of versican influences local and systemic tumor invasiveness in pre-clinical murine models. However, the mechanism(s) by which G3 influences breast tumor growth and metastasis is not well characterized. Here we evaluated the expression of versican in mouse mammary tumor cell lines observing that 4T1 cells expressed highest levels while 66c14 cells expressed low levels. We exogenously expressed a G3 construct in 66c14 cells and analyzed its effects on cell proliferation, migration, cell cycle progression, and EGFR signaling. Experiments in a syngeneic orthotopic animal model demonstrated that G3 promoted tumor growth and systemic metastasis in vivo. Activation of pERK correlated with high levels of G3 expression. In vitro, G3 enhanced breast cancer cell proliferation and migration by up-regulating EGFR signaling, and enhanced cell motility through chemotactic mechanisms to bone stromal cells, which was prevented by inhibitor AG 1478. G3 expressing cells demonstrated increased CDK2 and GSK-3β (S9P) expression, which were related to cell growth. The activity of G3 on mouse mammary tumor cell growth, migration and its effect on spontaneous metastasis to bone in an orthotopic model was modulated by up-regulating the EGFR-mediated signaling pathway. Taken together, EGFR-signaling appears to be an important pathway in versican G3-mediated breast cancer tumor invasiveness and metastasis.     

Doubled-haploid production in chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.): role of stress treatments

This is the first report on the production of double-haploid chickpea embryos and regenerated plants through anther culture using Canadian cultivar CDC Xena (kabuli) and Australian cultivar Sonali (desi). Maximum anther induction rates were 69% for Sonali and 63% for CDC Xena. Under optimal conditions, embryo formation occurred within 15–20 days of culture initiation with 2.3 embryos produced per anther for CDC Xena and 2.0 embryos per anther for Sonali. For anther induction, the following stress treatments were used: (1) flower clusters were treated at 4°C for 4 days, (2) anthers were subjected to electric shock treatment of three exponentially decaying pulses of 50–400 V with 25 μF capacitance and 25 Ω resistance, (3) anthers were centrifuged at 168–1,509g for 2–15 min, and finally (4) anthers were cultured for 4 days in high-osmotic pressure (563 mmol) liquid medium. Anthers were then transferred to a solid embryo development medium and, 15–20 days later, embryo development was observed concomitant with a small amount of callus growth of 0.1–3 mm. Anther-derived embryos were regenerated on plant regeneration medium. Electroporation treatment of anthers enhanced root formation, which is often a major hurdle in legume regeneration protocols. Cytological studies using DAPI staining showed a wide range of ploidy levels from haploid to tetraploid in 10–30-day-old calli. Flow cytometric analysis of calli, embryos and regenerated plants showed haploid profiles and/or spontaneous doubling of the chromosomes during early regeneration stages.     

Paleolimnological reconstruction of the trophic state in Lake Balaton (Hungary) using Cladocera remains

The sediment of Lake Balaton (Hungary) provides important information about the lake’s history, particularly with regard to eutrophication. In this study, we used fossil pigment analysis and subfossil Cladocera remains preserved in a dated sediment core to identify trophic stages from ~250 bc to present. Dates of the most recent eutrophic events are in good agreement with previously published data. In general, the abundance and diversity of the Cladocera community increased with eutrophication and decreased with oligotrophication. The sediments of Lake Balaton were characterised by Chydoridae remains, of which Alona species were the most abundant. Of these, Alona quadrangularis and Alona affinis accounted for 40 and 20% of the total Cladocera remains, respectively. The trophic state of Lake Balaton varied between mesotrophic and eutrophic regimes. Seven different trophic periods were identified in Lake Balaton on the basis of Sedimentary Pigment Degradation Unit (SPDU) content of the sediment. Eutrophic states were (1) from ~250 to ~30 bc, (3) between ~300 and ~590 ad, (5) between 1834 and 1944 and (7) from the 1960s until present. Mesotrophic states were (2) ~30 bc to ~300 ad, (4) 590–1834, (6) 1944–1960s. Discriminant analysis of the cladoceran data confirmed these historic events, except for the short mesotrophic episode between 1944 and 1960. The first stage of eutrophication of Lake Balaton (~250 to ~30 bc) was characterised by extensive macrophyte vegetation, as indicated by the increasing abundance of vegetation-associated Cladocera species (Eurycercus lamellatus, Sida crystallina, Pleuroxus sp.). Intensification of eutrophication was identified since the 1980s, reflected by a high abundance of Bosmina species. The most significant planktivorous fish of Lake Balaton was the Sabre carp (Pelecus cultratus), and when its number decreased, the abundance of Bosmina species increased. This study shows that Cladocera are responsive to trophic state changes, underlining their importance as a tool for the assessment of lake eutrophication.     

Inhibition of cGMP-dependent protein kinase II by its own splice isoform

cGMP- and cAMP-dependent protein kinases (cGK I, cGK II, and cAK) are important mediators of many signaling pathways that increase cyclic nucleotide concentrations and ultimately phosphorylation of substrates vital to cellular functions. Here we demonstrate a novel mRNA splice isoform of cGK II arising from alternative 5' splicing within exon 11. The novel splice variant encodes a protein (cGK II Delta(441-469)) lacking 29 amino acids of the cGK II Mg-ATP-binding/catalytic domain, including the conserved glycine-rich loop consensus motif Gly-x-Gly-x-x-Gly-x-Val which interacts with ATP in the protein kinase family of enzymes. cGK II Delta(441-469) has no intrinsic enzymatic activity itself, however, it antagonizes cGK II and cGK I, but not cAK. Thus, the activation and cellular functions of cGK II may be determined not only by intracellular cGMP levels but also by alternative splicing which may regulate the balance of expression of cGK II versus its own inhibitor, cGK II Delta(441-469).     

Critical role of mitochondrial glutathione in the survival of hepatocytes during hypoxia

Hypoxia is known to stimulate reactive oxygen species (ROS) generation. Because reduced glutathione (GSH) is compartmentalized in cytosol and mitochondria, we examined the specific role of mitochondrial GSH (mGSH) in the survival of hepatocytes during hypoxia (5% O2). 5% O2 stimulated ROS in HepG2 cells and cultured rat hepatocytes. Mitochondrial complex I and II inhibitors prevented this effect, whereas inhibition of nitric oxide synthesis with Nomega-nitro-L-arginine methyl ester hydrochloride or the peroxynitrite scavenger uric acid did not. Depletion of GSH stores in both cytosol and mitochondria enhanced the susceptibility of HepG2 cells or primary rat hepatocytes to 5% O2 exposure. However, this sensitization was abrogated by preventing mitochondrial ROS generation by complex I and II inhibition. Moreover, selective mGSH depletion by (R,S)-3-hydroxy-4-pentenoate that spared cytosol GSH levels sensitized rat hepatocytes to hypoxia because of enhanced ROS generation. GSH restoration by GSH ethyl ester or by blocking mitochondrial electron flow at complex I and II rescued (R,S)-3-hydroxy-4-pentenoate-treated hepatocytes to hypoxia-induced cell death. Thus, mGSH controls the survival of hepatocytes during hypoxia through the regulation of mitochondrial generation of oxidative stress.     

Activation of AMPK alpha- and gamma-isoform complexes in the intact ischemic rat heart

AMP-activated protein kinase (AMPK) plays a key role in modulating cellular metabolic processes. AMPK, a serine-threonine kinase, is a heterotrimeric complex of catalytic alpha-subunits and regulatory beta- and gamma-subunits with multiple isoforms. Mutations in the cardiac gamma(2)-isoform have been associated with hypertrophic cardiomyopathy and pre-excitation syndromes. However, physiological regulation of AMPK complexes containing different subunit isoforms is not well defined and is important for an understanding of the function of this signaling pathway in the intact heart. We evaluated the kinase activity associated with heart AMPK complexes containing specific alpha- and gamma-subunit isoforms of AMPK in an in vivo rat model of regional ischemia. Left coronary artery occlusion activated the immunoprecipitated alpha(1)-isoform (6-fold, P < 0.01) and alpha(2)-isoform (9-fold, P < 0.01) in the ischemic left ventricle compared with sham controls. The degree of alpha-subunit activation depended on the extent of ischemia and paralleled echocardiographic contractile dysfunction. The regulatory gamma(1)- and gamma(2)-isoforms were expressed in the heart. The gamma(1)- and gamma(2)-isoforms coimmunoprecipitated with alpha(1)- and alpha(2)-isoforms in proportion to alpha-subunit content. gamma(1)-Isoform immunocomplexes accounted for 70% of AMPK activity and AMPK phosphorylation (Thr(172)) in hearts. Ischemia similarly increased AMPK activity associated with the gamma(1)- and gamma(2)-isoform complexes threefold (P < 0.01 for each). Thus AMPK catalytic alpha(1)- and alpha(2)-isoforms are activated by regional ischemia in vivo in the heart, irrespective of the regulatory gamma(1)- or gamma(2)-isoforms to which they are complexed. Despite the pathophysiological importance of gamma(2)-isoform mutations, gamma(1)-isoform complexes account for most of the AMPK activity in the ischemic heart.     

Blood–Brain Transfer of Glucose and Glucose Analogs in Newborn Rats

Little is known of the selectivity of the blood-brain barrier at birth. Hexoses are transported through the barrier by a facilitating mechanism. To study the capacity of this mechanism to distinguish between analogs of D-glucose, we compared the transport of fluorodeoxyglucose, deoxyglucose, glucose, methylglucose, mannose, galactose, mannitol, and iodoantipyrine across the cerebral capillary endothelium in newborn Wistar rats. Cerebral blood flow, glucose consumption, and the blood-brain permeabilities of the hexoses were 25-50% of the adult values but the ratios between the permeabilities of the individual hexoses were similar to the ratios observed in adult rats. The mannitol clearance into brain was considerably higher than in adult rats (about 10-fold), indicating a higher endothelial permeability to small polar nonelectrolytes. The brain water content was higher in newborn than in adult rats and was associated with a higher steady-state distribution of labeled methylglucose between brain and blood. Hexose concentrations were determined relative to whole blood because the apparent erythrocyte membrane permeability to glucose was as high as in humans and thus considerably higher than in adult rats. The half-saturation concentration of glucose transport across the blood-brain barrier was considerably higher than in adult rats, about three-fold, suggesting that net blood-brain glucose transfer is less sensitive to blood glucose fluctuation in newborn than in adult rats.