Primary reactions of the LOV2 domain of phototropin,a plant blue-light photoreceptor

The phototropins constitute an important class of plant photoreceptor kinases that control a range of physiological responses, including phototropism, light-directed chloroplast movement, and light-induced stomatal opening. The LOV2 domain of phototropin binds a molecule of flavin mononucleotide (FMN) and undergoes a photocycle involving light-driven covalent adduct formation between a conserved cysteine residue and the C(4a) atom of FMN. This product state promotes C-terminal kinase activation and downstream signal transduction. Here, we report the primary photophysics and photochemistry of LOV2 domains of phototropin 1 of Avena sativa (oat) and of the phy3 photoreceptor of Adiantum capillus-veneris (maidenhair fern). In agreement with earlier reports [Swartz, T. E., et al. (2001) J. Biol. Chem. 276, 36493-36500], we find that the FMN triplet state is the reactive species from which the photoreaction occurs. We demonstrate that the triplet state is the primary photoproduct in the LOV2 photocycle, generated at 60% efficiency. No spectroscopically distinguishable intermediates precede the FMN triplet on the femtosecond to nanosecond time scale, indicating that it is formed directly via intersystem crossing (ISC) from the singlet state. Our results indicate that the majority of the FMN triplets in the LOV2 domain exist in the protonated form. We propose a reaction mechanism that involves excited-state proton transfer, on the nanosecond time scale or faster, from the sulfhydryl group of the conserved cysteine to the N5 atom of FMN. This event promotes adduct formation by increasing the electrophilicity of C(4a) and subsequent nucleophilic attack by the cysteine's thiolate anion. Comparison to free FMN in solution shows that the protein environment of LOV2 increases the ISC rate of FMN by a factor of 2.4, thus improving the yield of the cysteinyl-flavin adduct and the efficiency of phototropin-mediated signaling processes.     

Drosophila perlecan modulates FGF and hedgehog signals to activate neural stem cell division

Mutations in the Drosophila trol gene cause cell cycle arrest of neuroblasts in the larval brain. Here, we show that trol encodes the Drosophila homolog of Perlecan and regulates neuroblast division by modulating both FGF and Hh signaling. Addition of human FGF-2 to trol mutant brains in culture rescues the trol proliferation phenotype, while addition of a MAPK inhibitor causes cell cycle arrest of the regulated neuroblasts in wildtype brains. Like FGF, Hh activates stem cell division in the larval brain in a Trol-dependent fashion. Coimmunoprecipitation studies are consistent with interactions between Trol and Hh and between mammalian Perlecan and Shh that are not competed with heparin sulfate. Finally, analyses of mutations in trol, hh, and ttv suggest that Trol affects Hh movement. These results indicate that Trol can mediate signaling through both of the FGF and Hedgehog pathways to control the onset of stem cell proliferation in the developing nervous system.     

T3JAM, a novel protein that specifically interacts with TRAF3 and promotes the activation of JNK(1)

Previous studies suggest that localization of tumor necrosis factor receptor (TNFR)-associated factor (TRAF) family members is important for regulating their signal transduction. During a screen for TRAF3-associated proteins that potentially alter TRAF3 subcellular localization and enable signal transduction, we identified a novel protein, T3JAM (TRAF3-interacting Jun N-terminal kinase (JNK)-activating modulator). This protein associates specifically with TRAF3 but not other TRAF family members. Coexpression of T3JAM with TRAF3 recruits TRAF3 to the detergent-insoluble fraction. More importantly, T3JAM and TRAF3 synergistically activate JNK but not nuclear factor (NF)-kappaB. Our studies indicate that T3JAM may function as an adapter molecule that specifically regulates TRAF3-mediated JNK activation.     

Fasciola hepatica cathepsin L-like proteases: biology,function, and potential in the development of first generation liver fluke vaccines

Fasciola hepatica secretes cathepsin L proteases that facilitate the penetration of the parasite through the tissues of its host, and also participate in functions such as feeding and immune evasion. The major proteases, cathepsin L1 (FheCL1) and cathepsin L2 (FheCL2) are members of a lineage that gave rise to the human cathepsin Ls, Ks and Ss, but while they exhibit similarities in their substrate specificities to these enzymes they differ in having a wider pH range for activity and an enhanced stability at neutral pH. There are presently 13 Fasciola cathepsin L cDNAs deposited in the public databases representing a gene family of at least seven distinct members, although the temporal and spatial expression of each of these members in the developmental stage of F. hepatica remains unclear. Immunolocalisation and in situ hybridisation studies, using antibody and DNA probes, respectively, show that the vast majority of cathepsin L gene expression is carried out in the epithelial cells lining the parasite gut. Within these cells the enzyme is packaged into secretory vesicles that release their contents into the gut lumen for the purpose of degrading ingested host tissue and blood. Liver flukes also express a novel multi-domain cystatin that may be involved in the regulation of cathepsin L activity. Vaccine trials in both sheep and cattle with purified native FheCL1 and FheCL2 have shown that these enzymes can induce protection, ranging from 33 to 79%, to experimental challenge with metacercariae of F. hepatica, and very potent anti-embryonation/hatch rate effects that would block parasite transmission. In this article we review the vaccine trials carried out over the past 8 years, the role of antibody and T cell responses in mediating protection and discuss the prospects of the cathepsin Ls in the development of first generation recombinant liver fluke vaccines.     

A katanin-like protein regulates normal cell wall biosynthesis and cell elongation

Fibers are one of the mechanical tissues that provide structural support to the plant body. To understand how the normal mechanical strength of fibers is regulated, we isolated an Arabidopsis fragile fiber (fra2) mutant defective in the mechanical strength of interfascicular fibers in the inflorescence stems. Anatomical and chemical analyses showed that the fra2 mutation caused a reduction in fiber cell length and wall thickness, a decrease in cellulose and hemicellulose contents, and an increase in lignin condensation, indicating that the fragile fiber phenotype of fra2 is a result of alterations in fiber cell elongation and cell wall biosynthesis. In addition to the effects on fibers, the fra2 mutation resulted in a remarkable reduction in cell length and an increase in cell width in all organs, which led to a global alteration in plant morphology. The FRA2 gene was shown to encode a protein with high similarity to katanin (hence FRA2 was renamed AtKTN1), a protein shown to be involved in regulating microtubule disassembly by severing microtubules. Consistent with the putative function of AtKTN1 as a microtubule-severing protein, immunolocalization demonstrated that the fra2 mutation caused delays in the disappearance of perinuclear microtubule array and in the establishment of transverse cortical microtubule array in interphase and elongating cells. Together, these results suggest that AtKTN1, a katanin-like protein, is essential not only for normal cell wall biosynthesis and cell elongation in fiber cells but also for cell expansion in all organs.     

Regulation of secretory granule pH in insulin-secreting cells

Luminal acidification is important for the maturation of secretory granules, yet little is known regarding the regulation of pH within them. A pH-sensitive green fluorescent protein (EGFP) was targeted to secretory granules in RIN1046-38 insulinoma cells by using a construct in which the EGFP gene was preceded by the nucleotide sequence for human growth hormone. Stimulatory levels of glucose doubled EGFP secretion from cell cultures, and potentiators of glucose-induced insulin secretion enhanced EGFP release. Thus this targeted EGFP is useful for population measurements of secretion. However, less than ~4% of total cell EGFP was released after 1.5 h of stimulation. Consequently, when analyzed in single cells, fluorescence of the targeted EGFP acts as an indicator of pH within secretory granules. Glucose elicited a decrease in granule pH, whereas inhibitors of the V-type H(+)-ATPase increased pH and blocked the glucose effect. Granule pH also was modified by effectors of the protein kinase A pathway, with activation eliciting granule alkalinization, suggesting that potentiation of peptide release by cAMP may involve regulated changes in secretory granule pH.     

Interactive effects of lateral shade and wind on stem allometry, biomass allocation, and mechanical stability in Abutilon theophrasti (Malvaceae)

The effects of lateral shade and wind on stem allometry, whole-plant biomass allocation, and mechanical stability were examined for Abutilon theophrasti in a fully factorial glasshouse experiment. Lateral shade from neighboring plants increased stem height by 33% relative to control plants grown individually, despite a decrease in plant dry mass. Intermittent wind decreased stem height by 18% in unshaded plants, but by only 3% in shaded plants. Surprisingly, both lateral shade and wind caused decreases in stem diameter, even with diameter controlled for height, resulting in low diameter?:?height ratios in wind-treated plants relative to untreated plants. Under shade, wind-treated plants had higher root allocation than untreated plants, which allowed wind-treated shade plants to compensate for a low diameter?:?height ratio. This did not occur in the absence of shade, where stem tissue density and root allocation of wind-treated plants did not exceed that of untreated plants. Nevertheless, wind-treated plants experienced low drag relative to untreated plants due to a lower leaf area. Consequently, stem deflections of wind-treated plants did not exceed those of untreated plants at any given windspeed. Our results document a complex interaction between shade and wind on plant morphology and suggest that the nature of this interaction is generally that lateral shade acts to reduce or eliminate thigmomorphogenic responses.     

The relationship between ligand aggregation and G-quadruplex DNA selectivity in a series of 3,4,9,10-perylenetetracarboxylic acid diimides

Human telomeres are comprised of d(TTAGGG) repeats that are capable of forming G-quadruplex DNA structures. Ligands that bind to and stabilize these G-quadruplex DNA structures are potential inhibitors of the cancer cell-associated enzyme telomerase. Other potential biological uses of G-quadruplex targeting ligands have been proposed. One particularly challenging aspect of the contemplated uses of G-quadruplex targeting ligands is their selectivity for G-quadruplex DNA versus double-stranded DNA structures. We have previously reported the observation that two structurally related 3,4,9,10-perylenetetracarboxylic acid diimide-based G-quadruplex DNA ligands, PIPER [N,N'-bis(2-(1-piperidino)ethyl)-3,4,9,10-perylenetetracarboxylic acid diimide] and Tel01 [N,N'-bis(3-(4-morpholino)propyl)-3,4,9,10-perylenetetracarboxylic acid diimide], have different levels of G-quadruplex DNA binding selectivity at pH 7 as determined by absorbance changes in the presence of different DNA structures [Kerwin, S. M., Chen, G., Kern, J. T., and Thomas, P. W. (2002) Bioorg. Med. Chem. Lett. 12, 447-450]. Here we report that the less G-quadruplex DNA selective ligand PIPER can unwind double-stranded, closed circular plasmid DNA, as determined by a topoisomerase I assay. A model for the interaction of Tel01 with the G-quadruplex DNA structure formed by d(TAGGGTTA) was determined from NMR experiments. This model is similar to the previously published model for PIPER bound to the same G-quadruplex DNA and failed to provide a structural basis for the observed increased selectivity of Tel01 interaction with G-quadruplex DNA. In contrast, investigation into the aggregation state of Tel01 and PIPER as well as other 3,4,9,10-perylenetetracarboxylic acid diimide analogues bearing basic side chains demonstrates that ligand aggregation is correlated with G-quadruplex DNA binding selectivity. For all six analogues examined, those ligands that were aggregated at pH 7 in 70 mM potassium phosphate, 100 mM KCl, 1 mM EDTA buffer also demonstrated G-quadruplex DNA binding selectivity under these buffer conditions. Ligands that were not aggregated under these conditions display much lower levels of G-quadruplex DNA selectivity. The aggregation state of these ligands is extremely sensitive to the buffer pH. Tel01, which is aggregated at pH 7, is not aggregated at pH 6.4, where it demonstrates only modest G-quadruplex DNA binding selectivity, and PIPER in pH 8.5 buffer is both aggregated and highly G-quadruplex DNA-selective. To our knowledge, these studies demonstrate the first DNA structure selectivity as achieved through pH-mediated ligand aggregation. The potential impact of these findings on the selectivity of other classes of G-quadruplex DNA ligands is discussed.     

Diagnosis of gastric glomus tumor by endoscopic ultrasound-guided fine needle aspiration biopsy. A case report with cytologic,histologic and immunohistochemical studies

BACKGROUND: The majority of glomus tumor are small, benign neoplasms that arise from modified smooth muscle cells. They usually occur in the dermis or subcutis of the extremities. However, rare cases have been reported in the visceral locations, most often in the stomach. CASE: A 32-year-old woman presented with episodes of right upper quadrant pain. She was found to have a gastric tumor that was biopsied at another hospital, where the diagnosis of gastrointestinal stromal tumor (GIST) was made. Endoscopic ultrasound (EUS) performed at our institution revealed a gastric submucosal tumor that was then biopsied by fine needle aspiration (FNA). Cytology revealed cohesive clusters of uniform, round, small cells with ill-defined cytoplasmic borders and scanty, amphophilic cytoplasm. Nuclei were round, with smooth nuclear membranes and evenly distributed, dusty chromatin. Intermingled with those epithelioid cells were small, short, spindled, normal endothelial cells. Immunohistochemical studies performed on cell block showed that the tumor cells were negative for CD34, CD117, chromogranin, synaptophysin, desmin and AE1/AE3 and were strongly positive for SMA, HHF-35 and collagen type IV. Glomus tumor was diagnosed and later confirmed by histology. CONCLUSION: EUS-guided FNA biopsy is efficient and permits adequate sampling for accurate diagnosis of gastric glomus tumor. Although rare, glomus tumor should be in the differential diagnosis among other gastric lesions, such as well-differentiated adenocarcinoma, epithelioid GIST and carcinoid tumor.