Three RNA recognition motifs participate in RNA recognition and structural organization by the pro-apoptotic factor TIA-1

T-cell intracellular antigen-1 (TIA-1) regulates developmental and stress-responsive pathways through distinct activities at the levels of alternative pre-mRNA splicing and mRNA translation. The TIA-1 polypeptide contains three RNA recognition motifs (RRMs). The central RRM2 and C-terminal RRM3 associate with cellular mRNAs. The N-terminal RRM1 enhances interactions of a C-terminal Q-rich domain of TIA-1 with the U1-C splicing factor, despite linear separation of the domains in the TIA-1 sequence. Given the expanded functional repertoire of the RRM family, it was unknown whether TIA-1 RRM1 contributes to RNA binding as well as documented protein interactions. To address this question, we used isothermal titration calorimetry and small-angle X-ray scattering to dissect the roles of the TIA-1 RRMs in RNA recognition. Notably, the fas RNA exhibited two binding sites with indistinguishable affinities for TIA-1. Analyses of TIA-1 variants established that RRM1 was dispensable for binding AU-rich fas sites, yet all three RRMs were required to bind a polyU RNA with high affinity. Small-angle X-ray scattering analyses demonstrated a "V" shape for a TIA-1 construct comprising the three RRMs and revealed that its dimensions became more compact in the RNA-bound state. The sequence-selective involvement of TIA-1 RRM1 in RNA recognition suggests a possible role for RNA sequences in regulating the distinct functions of TIA-1. Further implications for U1-C recruitment by the adjacent TIA-1 binding sites of the fas pre-mRNA and the bent TIA-1 shape, which organizes the N- and C-termini on the same side of the protein, are discussed.     

Manipulation of <Emphasis Type="SmallCaps">l</Emphasis>-ascorbic acid biosynthesis pathways in <Emphasis Type="Italic">Solanum lycopersicum</Emphasis>: elevated GDP-mannose pyrophosphorylase activity enhances <Emphasis Type="SmallCaps">l</Emphasis>-ascorbate levels in red fruit

Ascorbate (AsA) plays a fundamental role in redox homeostasis in plants and animals, primarily by scavenging reactive oxygen species. Three genes, representing diverse steps putatively involved in plant AsA biosynthesis pathways, were cloned and independently expressed in Solanum lycopersicum (tomato) under the control of the CaMV 35S promoter. Yeast-derived GDP-mannose pyrophosphorylase (GMPase) and arabinono-1,4-lactone oxidase (ALO), as well as myo-inositol oxygenase 2 (MIOX2) from Arabidopsis thaliana, were targeted. Increases in GMPase activity were concomitant with increased AsA levels of up to 70% in leaves, 50% in green fruit, and 35% in red fruit. Expression of ALO significantly pulled biosynthetic flux towards AsA in leaves and green fruit by up to 54 and 25%, respectively. Changes in AsA content in plants transcribing the MIOX2 gene were inconsistent in different tissue. On the other hand, MIOX activity was strongly correlated with cell wall uronic acid levels, suggesting that MIOX may be a useful tool for the manipulation of cell wall composition. In conclusion, the Smirnoff–Wheeler pathway showed great promise as a target for biotechnological manipulation of ascorbate levels in tomato.     

Systematics and evolution of the Australian knob-tail geckos (Nephrurus, Carphodactylidae, Gekkota): plesiomorphic grades and biome shifts through the Miocene

Clades that predate the origin of biomes that they inhabit provide unique opportunities to examine both when major environmental transitions occurred, and how lineages adapted to these changes. The isolated island continent Australia has undergone a profound environmental transition through the Miocene, from relatively mesic to predominantly arid; however, we have much to learn about both the timing of this change, and how organisms may have responded to it. The family Carphodactylidae is an ancient Gondwanan group of geckos that occurs across all major Australian biomes. A multilocus (ND2, Rag-1, c-mos) phylogenetic and dating analysis of the most ecologically diverse clade within this group, the genus Nephrurus (sensuBauer, 1990) reveals that two of three morphological taxa historically recognized (the 'spiny knob-tails' and 'Underwoodisaurus') are relatively species depauperate, pleisomorphic basal grades that diversified through the late Oligocene and early Miocene, and are now absent from most of the arid biome. Based on their deep divergence and morphological distinctiveness we recognize two lineages (milii and sphyrurus) as monotypic genera, the later of which is named herein (Uvidicolus nov. gen). In contrast, a third morphological group, the 'smooth knob-tails,' is a monophyletic group of five exclusively arid zone burrowing species that has radiated relatively recently (mid-Miocene). Our phylogeny indicates that successful colonization of this novel and challenging biome by Nephrurus correlates with an initial shift to terrestriality and adaptation to at least seasonally arid conditions around the early Miocene, and the eventual evolution and subsequent mid-Miocene radiation of a lineage specialized for burrowing.     

Capsaicin Modulates Proliferation,Migration, and Activation of Hepatic Stellate Cells

Capsaicin, the active component of chili pepper, has been reported to have antiproliferative and anti-inflammatory effects on a variety of cell lines. In the current study, we aimed to investigate the effects of capsaicin during HSC activation and maintenance. Activated and freshly isolated HSCs were treated with capsaicin. Proliferation was measured by incorporation of EdU. Cell cycle arrest and apoptosis were investigated using flow cytometry. The migratory response to chemotactic stimuli was evaluated by a modified Boyden chamber assay. Activation markers and inflammatory cytokines were determined by qPCR, immunocytochemistry, and flow cytometry. Our results show that capsaicin reduces HSC proliferation, migration, and expression of profibrogenic markers of activated and primary mouse HSCs. In conclusion, the present study shows that capsaicin modulates proliferation, migration, and activation of HSC in vitro.     

A novel, non-invasive, online-monitoring, versatile and easy plant-based probe for measuring leaf water status

A high-precision pressure probe is described which allows non-invasive online-monitoring of the water relations of intact leaves. Real-time recording of the leaf water status occurred by data transfer to an Internet server. The leaf patch clamp pressure probe measures the attenuated pressure, P(p), of a leaf patch in response to a constant clamp pressure, P(clamp). P(p) is sensed by a miniaturized silicone pressure sensor integrated into the device. The magnitude of P(p) is dictated by the transfer function of the leaf, T(f), which is a function of leaf patch volume and ultimately of cell turgor pressure, P(c), as shown theoretically. The power function T(f)=f(P(c)) theoretically derived was experimentally confirmed by concomitant P(p) and P(c) measurements on intact leaflets of the liana Tetrastigma voinierianum under greenhouse conditions. Simultaneous P(p) recordings on leaflets up to 10 m height above ground demonstrated that changes in T(f) induced by P(c) changes due to changes of microclimate and/or of the irrigation regime were sensitively reflected in corresponding changes of P(p). Analysis of the data show that transpirational water loss during the morning hours was associated with a transient rise in turgor pressure gradients within the leaflets. Subsequent recovery of turgescence during the afternoon was much faster than the preceding transpiration-induced water loss if the plants were well irrigated. Our data show the enormous potential of the leaf patch clamp pressure probe for leaf water studies including unravelling of the hydraulic communication between neighbouring leaves and over long distances within tall plants (trees).     

Quantitative analysis of a nuclear antigen in interphase and mitotic cells

The quantification of an interchromatin-associated antigen, designated p 105, during cellular passage through mitosis is described. Indirect immunofluorescence microscopy and immunogold electron microscopy demonstrated a qualitative increase in p 105 within the mitotic cytoplasm. Multiparameter flow cytometric analysis was performed on fixed cells sequentially stained with anti-p 105 immunofluorescence and/or propidium iodide. This analysis demonstrated approximately a tenfold increase in intracellular p 105 content as a function of progression from the G2 to the M phase. This increase was corroborated by the quantitative immunoblot analysis of colchicine-treated cell cultures and of cells sorted on the basis of anti-p 105 immunofluorescence. The data reveal that the increased levels of anti-p 105 immunofluorescence in conjunction with flow cytometry may be used effectively to quantitate mitotic index and isolate mitotic cells. The function and modulation of p 105 throughout the cell cycle is discussed.     

Mutational analysis of the thyrotropin-releasing hormone-degrading ectoenzyme. similarities and differences with other members of the M1 family of aminopeptidases and thermolysin

Thyrotropin-releasing hormone-degrading ectoenzyme (TRH-DE) is a TRH-specific peptidase which catalyzes the inactivation of the peptidergic signal substance TRH. As indicated by sequence alignment, TRH-DE and the other members of the M1 family of aminopeptidases have a distinct set of conserved amino acid residues in common. By replacing amino acid residues that are putatively involved in catalysis, we could demonstrate that the enzymatic activities of the mutants E408D, E442D, E464Q, E464D, Y528F, H507R, and H507F are dramatically decreased, essentially due to the changes of V(max). The mutant enzymes E408Q and E442Q are inactive, whereas the specific enzymatic activity of the mutants R488Q, R488A, and Y554F are similar to that of the wild-type enzyme. These data strongly suggest that E408, E442, Y528, and H507 are involved in the catalytic process of TRH-DE while E464 presumably represents the third zinc-coordinating residue and may be equivalent to E166 in thermolysin. In contrast, amino acid residues R488 and Y554 seem not to be involved in the catalytic mechanism of TRH-DE.     

Exonuclease V from Saccharomyces cerevisiae. A 5'----3'-deoxyribonuclease that produces dinucleotides in a sequential fashion

A novel deoxyribonuclease, exonuclease V, has been purified approximately 30,000-fold from Saccharomyces cerevisiae. Exonuclease V is localized in the nucleus. The nuclease degrades single-stranded, but not double-stranded, DNA from the 5'-end. The products of exonuclease action are dinucleotides, except the 3'-terminal tri- and tetranucleotides which are not degraded. Studies with synthetic oligo- and polynucleotides with specified sequence elements showed that exonuclease V cleaves off dinucleotides as primary digestion products. Thus, the polymers (pT)9pA(pT)n and (pT)10pA(pT)n yielded pTpA and pApT as digestion products, respectively. Removal of the 5'-terminal phosphate from the DNA substrate results in reduced binding of the enzyme to the substrate. In addition, the initial hydrolytic cut by exonuclease V on the dephosphorylated substrate produces a mixture of dinucleoside monophosphates and trinucleoside diphosphates. The enzyme is processive in action.     

SMS 201-995: a very potent and selective octapeptide analogue of somatostatin with prolonged action

Stepwise modification of a conformationally stabilised analogue of the fragment of somatostatin which had been thought to be essential biologically active moiety has enabled us to synthesise the analogue H-(D) Phe-Cys-Phe-(D) Trp-Lys-Thr-Cys-Thr(ol) code-named SMS 201-995, which in vitro is three times more potent than the native hormone in inhibiting the secretion of growth hormone, which is highly resistant to degradation by pure enzymes and by tissue homogenates, which in vivo in rat and rhesus monkey is (depending on test system) at least 20 times more active than somatostatin, which is much longer acting, and which moreover in both species is much more selective in inhibiting the secretion of growth hormone than that of insulin. The compound is active by several routes of administration including the oral, is well tolerated both in laboratory animals and in man, and is currently undergoing preliminary clinical trial.