THEORY AND MEASUREMENT OF VISUAL MECHANISMS : VII. THE FLICKER RESPONSE FUNCTION OUTSIDE THE FOVEA

The several parameters of the flicker response contour (F – log I) are considered as a function of wave-length composition (white, blue, and red) and light-time fraction, for an extra-foveal region (monocular, temporal retina). These data are compared with those secured for the same image area centrally fixated at the fovea. The systematic changes in the parameters are shown to be in rational relation to other relevant excitability data. Since for two retinal regions the primary contours are quite different, the systematic nature of the behavior of the parameters in the two cases is a real test of the power of the analysis proposed. Theoretical interpretation is required to deal with the properties of sets of performance contours under systematically varied conditions, and cannot rely simply on the comparison of (for example) two contours under the same arbitrary conditions at two retinal locations. In particular it is emphasized that a qualitative separation must be made of the two factors of (a) number of units and (b) the frequencies of their actions, before the wave-length problem can be dealt with effectively.     

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.     

A gene of the major facilitator superfamily encodes a transporter for enterobactin (Enb1p) in Saccharomyces cerevisiae

While in fungi iron transport via hydroxamate siderophores has been amply proven, iron transport via enterobactin is largely unknown. Enterobactin is a catecholate-type siderophore produced by several enterobacterial genera grown in severe iron deprivation. By using the KanMX disruption module in vector pUG6 in a fet3 background of Saccharomyces cerevisiae we were able to disrupt the gene YOL158c Sce of the major facilitator super family (MFS) which has been previously described as a gene encoding a membrane transporter of unknown function. Contrary to the parental strain, the disruptant was unable to utilize ferric enterobactin in growth promotion tests and in transport assays using ⁵⁵Fe-enterobactin. All other siderophore transport properties remained unaffected. The results are evidence that in S. cerevisiae the YOL158c Sce gene of the major facilitator super family, now designated ENB1, encodes a transporter protein (Enb1p), which specifically recognizes and transports enterobactin.     

Four distinct chondrocyte populations in the fetal bovine growth plate: highest expression levels of PTH/PTHrP receptor,Indian hedgehog,and MMP-13 in hypertrophic chondrocytes and their suppression by PTH (1-34) and PTHrP (1-40)

Differentiation and growth of chondrocytes in fetal growth plates of vertebrate long bones and ribs appear to occur in a gradual, continuous manner between the resting zone through the proliferation zone, maturation zone, and upper and lower hypertrophic zones, with a continuous increase in cell size up to 10-fold of the volume of a resting chondrocyte. Here we provide evidence, however, that after centrifugation through a continuous Percoll gradient growth plate chondrocytes separate into four distinct cell populations (B1 to B4) which differ markedly in density, size, and gene expression. These populations collect in the absence of any phase borders in the gradient which might serve as concentration barriers. Fractions B1 and B2 contained the largest cells with the lowest buoyant density and showed the highest expression levels for type X collagen (Col X), but only the B1 population expressed high levels of matrix metalloproteinase-13 (collagenase 3). Cells in fraction B3 were significantly smaller and expressed little Col X, while cells in fraction B4 were of similar size to cells in the resting zone without significant Col X expression. The highest levels of parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor (PTHR-1), and Indian hedgehog (Ihh) expression were also found in the hypertrophic fractions B1 and B2 and not in the prehypertrophic fraction B3, as expected from in situ hybridization data on PTHR-1 expression in fetal rodent or chicken growth plates. Incubation of fractions B1 to B3 with the amino-terminal fragments PTH (1-34) or PTHrP (1-40) suppressed the expression of Col X and PTHR-1 by more than 50% and the expression of Ihh nearly completely. In contrast, the mid-regional PTH fragment PTH (28-48) and PTH (52-84) consistently stimulated the expression of PTHR-1 by 10-20% in fractions B1 to B3. These findings confirm the existence of distinct differentiation stages within chondrocytes of the growth plate and support the hypothesis proposed by Vortkamp et al. (Science 273(1996)613) of a regulatory feedback loop of Ihh and PTH/PTHrP fragments controlling the differentiation of proliferating to prehypertrophic chondrocytes, but extend the ability to respond to PTH/PTHrP hypertrophic chondrocytes.