Photosynthetic Gas Exchange and Discrimination against 13CO2 and C18O16O in Tobacco Plants Modified by an Antisense Construct to Have Low Chloroplastic Carbonic Anhydrase

The physiological role of chloroplastic carbonic anhydrase (CA) was examined by antisense suppression of chloroplastic CA (on average 8% of wild type) in Nicotiana tabacum. Photosynthetic gas-exchange characteristics of low-CA and wild-type plants were measured concurrently with short-term, on-line stable isotope discrimination at varying vapor pressure deficit (VPD) and light intensity. Low-CA and wild-type plants were indistinguishable in the responses of assimilation, transpiration, stomatal conductance, and intercellular CO2 concentration to changing VPD or light intensity. At saturating light intensity, low-CA plants had lower discrimination against 13CO2 than wild-type plants by 1.2 to 1.8[per mille (thousand) sign]. Consequently, tissue of the low-CA plants was higher in 13C than the control plants. It was calculated that low-CA plants had chloroplast CO2 concentrations 13 to 22 [mu]mol mol-1 lower than wild-type plants. Discrimination against C18O16O in low-CA plants was 20% of that of the wild type, confirming a role of chloroplastic CA in the mechanism of discrimination against C18O16O ([delta]C18O16O). As VPD increased, stomatal closure caused a reduction in chloroplastic C02 concentration, and since VPD and chloroplastic CO2 concentration act in opposing directions on [delta]C18O16O, no effect of VPD was seen on [delta]C18O16O.     

Discovery of pyrazolo[1,5-a]pyridines as p110α-selective PI3 kinase inhibitors

We have made a novel series of pyrazolo[1,5-a]pyridines as PI3 kinase inhibitors, and demonstrated their selectivity for the p110α isoform over the other Class Ia PI3 kinases. We investigated the SAR around the pyrazolo[1,5-a]pyridine ring system, and found compound 5x to be a particularly potent example (p110α IC(50) 0.9nM). This compound inhibits cell proliferation and phosphorylation of Akt/PKB, a downstream marker of PI3 kinase activity, and showed in vivo activity in an HCT-116 human xenograft model.     

Atractis marquezi n. sp. (Nematoda: Atractidae) and a revision of Atractis dujardin, 1845, sensu Baker, 1987

Atractis marquezi n. sp. from the large intestine of the tortoise Geochelone nigrita is described and illustrated. Atractis Dujardin, 1845, sensu Baker, 1987, is revised to contain only those atractids having a lagonoid spicule. The remaining species of Atractis sensu Baker, 1987, are assigned to Cyrtosomum, Pseudatractis, and Klossinemella. A. marquezi represents the 12th species to be assigned to this genus and is distinguished from other Neotropical species by the distribution pattern of caudal papillae of the male: 2 pairs precloacal, 2 pairs adcloacal, and 6 pairs postcloacal.     

Cysteine-disulfide cross-linking to monitor SNARE complex assembly during endoplasmic reticulum-Golgi transport

Assembly of cognate SNARE proteins into SNARE complexes is required for many intracellular membrane fusion reactions. However, the mechanisms that govern SNARE complex assembly and disassembly during fusion are not well understood. We have devised a new in vitro cross-linking assay to monitor SNARE complex assembly during fusion of endoplasmic reticulum (ER)-derived vesicles with Golgi-acceptor membranes. In Saccharomyces cerevisiae, anterograde ER-Golgi transport requires four SNARE proteins: Sec22p, Bos1p, Bet1p, and Sed5p. After tethering of ER-derived vesicles to Golgi-acceptor membranes, SNARE proteins are thought to assemble into a four-helix coiled-coil bundle analogous to the structurally characterized neuronal and endosomal SNARE complexes. Molecular modeling was used to generate a structure of the four-helix ER-Golgi SNARE complex. Based on this structure, cysteine residues were introduced into adjacent SNARE proteins such that disulfide bonds would form if assembled into a SNARE complex. Our initial studies focused on disulfide bond formation between the SNARE motifs of Bet1p and Sec22p. Expression of SNARE cysteine derivatives in the same strain produced a cross-linked heterodimer of Bet1p and Sec22p under oxidizing conditions. Moreover, this Bet1p-Sec22p heterodimer formed during in vitro transport reactions when ER-derived vesicles containing the Bet1p derivative fused with Golgi membranes containing the Sec22p derivative. Using this disulfide cross-linking assay, we show that inhibition of transport with anti-Sly1p antibodies blocked formation of the Bet1p-Sec22p heterodimer. In contrast, chelation of divalent cations did not inhibit formation of the Bet1p-Sec22p heterodimer during in vitro transport but potently inhibited Golgi-specific carbohydrate modification of glyco-pro-alpha factor. This data suggests that Ca(2+) is not directly required for membrane fusion between ER-derived vesicles and Golgi-acceptor membranes.     

The stable carbon and nitrogen isotopic composition of vegetation in tropical forests of the Amazon Basin, Brazil

Here we present the within-site, seasonal, and interannual variations of the carbon (δ¹³C) and nitrogen (δ¹⁵N) isotope ratios of leaves, wood, bark and litter from four sites in the Amazon region, Brazil. Samples were collected in Manaus (3° 06′07′′ S; 60°01′30′′ W), Ji-Paraná (10°53′07′′ S; 61°57′06′′ W), and Santarém (2°26′35′′ S; 54°42′30′′ W) with mean annual precipitation of 2207, 2040 and 1909 mm respectively. The overall average for all leaf samples was for δ¹³C and for δ¹⁵N (n=756). The leaf δ values at these sites were often but not always statistically distinct from each other. The δ¹³C values varied from to . Pronounced differences in δ¹³C values occurred with height associated with differences in forest structure. The δ¹³C of leaf dry matter showed seasonal variations associated with the length of the dry season, despite the fact that total annual precipitation was similar among the studied sites. Leaf δ¹⁵N values ranged from to a maximum value of , and the Santarém sites showed more enriched values than Manaus and Ji-Paraná sites. No seasonal variation was detected in the δ¹⁵N of leaves, but significant differences were observed among sites and with changes in canopy height. The isotope ratio data are consistent with our current understanding of the roles of light, water availability, and recycling of soil-respired CO₂ influences on δ¹³C and consistent with our understanding that an open nitrogen cycle can lead to high δ¹⁵N values despite a significant number of legumes in the vegetation.     

Human glutathione transferase T2-2 discloses some evolutionary strategies for optimization of substrate binding to the active site of glutathione transferases

Rapid kinetic, spectroscopic, and potentiometric studies have been performed on human Theta class glutathione transferase T2-2 to dissect the mechanism of interaction of this enzyme with its natural substrate GSH. Theta class glutathione transferases are considered to be older than Alpha, Pi, and Mu classes in the evolutionary pathway. As in the more recently evolved GSTs, the activation of GSH in the human Theta enzyme proceeds by a forced deprotonation of the sulfhydryl group (pK(a) = 6.1). The thiol proton is released quantitatively in solution, but above pH 6.5, a protein residue acts as an internal base. Unlike Alpha, Mu, and Pi class isoenzymes, the GSH-binding mechanism occurs via a simple bimolecular reaction with k(on) and k(off) values at least hundred times lower (k(on) = (2.7 +/- 0.8) x 10(4) M(-1) s(-1), k(off) = 36 +/- 9 s(-1), at 37 degrees C). Replacement of Arg-107 by alanine, using site-directed mutagenesis, remarkably increases the pK(a) value of the bound GSH and modifies the substrate binding modality. Y107A mutant enzyme displays a mechanism and rate constants for GSH binding approaching those of Alpha, Mu, and Pi isoenzymes. Comparison of available crystallographic data for all these GSTs reveals an unexpected evolutionary trend in terms of flexibility, which provides a basis for understanding our experimental results.     

An automated image capture and quantitation approach to identify proteins affecting tumor cell proliferation

To facilitate the characterization of proteins that negatively regulate tumor cell proliferation in vitro, the authors have implemented a high-throughput functional assay that measures S-phase progression of tumor cell lines. For 2 tumor cell lines-human melanoma A375 and human lung carcinoma A549-conditions were established using the cyclin-dependent kinase inhibitor, p27kip; the tumor suppressor p53, a kinase-inactive allele of the cell cycle-regulated serine/threonine kinase Aurora2; and the G1/S drug block, aphidicolin. For screening purposes, gene libraries were delivered by adenoviral infection. Cells were fixed and labeled by immunocytochemistry, and an automated image acquisition and analysis package on a Cellomics ArrayScanII was used to quantify the effects of these treatments on cell proliferation. The assay can be used to identify novel proteins involved in proliferation and serves as a more robust, reproducible, and sensitive alternative to enzyme-linked immunosorbent assay (ELISA)-based technologies.     

Axonal protein synthesis provides a mechanism for localized regulation at an intermediate target

As axons grow past intermediate targets, they change their responsiveness to guidance cues. Local upregulation of receptor expression is involved, but the mechanisms for this are not clear. Here protein synthesis is traced within individual axons by introducing RNAs encoding visualizable reporters. Individual severed axons and growth cones can translate proteins and also export them to the cell surface. As axons reach the spinal cord midline, EphA2 is among the receptors upregulated on at least some distal axon segments. Midline reporter upregulation is recapitulated by part of the EphA2 mRNA 3' untranslated region, which is highly conserved and includes known translational control sequences. These results show axons contain all the machinery for protein translation and cell surface expression, and they reveal a potentially general and flexible RNA-based mechanism for regulation localized within a subregion of the axon.     

Reversible G(1) arrest induced by inhibition of the epidermal growth factor receptor tyrosine kinase requires up-regulation of p27(KIP1) independent of MAPK activity

We have used quinazoline inhibitors of the epidermal growth factor receptor (EGFR) tyrosine kinase to study the link between EGFR signaling and G(1) to S traverse. Treatment of A431 and MDA-468 human tumor cells with 0.1-10 microM AG-1478 inhibited basal and ligand-stimulated EGFR phosphorylation without a decrease in receptor content, EGF-binding sites, or binding affinity. Incubation of A431 cells with 0.1-1 microM AG-1517 abrogated (125)I-EGF internalization. Both AG-1478 and AG-1517 markedly inhibited A431 and MDA-468 colony formation in soft agarose at concentrations between 0.01 and 1 microM. Daily injections of AG-1478 at 50 mg/kg delayed A431 tumor formation in athymic nude mice. A transient exposure of A431 cells to AG-1478 resulted in a dose-dependent up-regulation of the cyclin-dependent kinase inhibitor p27, down-regulation of cyclin D1 and of active MAPK, and hypophosphorylation of the retinoblastoma protein (Rb). These changes were temporally associated with recruitment of tumor cells in G(1) phase and a marked reduction of the proportion of cells in S phase. Upon removal of the kinase inhibitor, EGFR and Rb phosphorylation and the levels of cyclin D1 protein were quickly restored, but the cells did not reenter S phase until p27 protein levels were decreased. Phosphorothioate p27 oligonucleotides decreased p27 protein in A431 cells and abrogated the quinazoline-mediated G(1) arrest. Treatment of A431 cells with PD 098509, a synthetic inhibitor of MEK1, inhibited MAPK activity without inducing G(1) arrest or increasing the levels of p27. However, treatment with LY 294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K), inhibited basal Akt activity, up-regulated p27, and recruited cells in G(1). These data suggest that p27 is required for the growth arrest that follows interruption of the EGFR kinase in receptor-overexpressing cells. In addition, the G(1) arrest and up-regulation of p27 resulting from EGFR blockade are not due to the interruption of MAPK, but to the interruption of constitutively active PI3K function.