Intracellular sensing of amino acids in Xenopus laevis oocytes stimulates p70 S6 kinase in a target of rapamycin-dependent manner.

Amino acids exert modulatory effects on proteins involved in control of mRNA translation in animal cells through the target of rapamycin (TOR) signaling pathway. Here we use oocytes of Xenopus laevis to investigate mechanisms by which amino acids are "sensed" in animal cells. Small ( approximately 48%) but physiologically relevant increases in intracellular but not extracellular total amino acid concentration (or Leu or Trp but not Ala, Glu, or Gln alone) resulted in increased phosphorylation of p70(S6K) and its substrate ribosomal protein S6. This response was inhibited by rapamycin, demonstrating that the effects require the TOR pathway. Alcohols of active amino acids substituted for amino acids with lower efficiency. Oocytes were refractory to changes in external amino acid concentration unless surface permeability of the cell to amino acids was increased by overexpression of the System L amino acid transporter. Amino acid-induced, rapamycin-sensitive activation of p70(S6K) was conferred when System L-expressing oocytes were incubated in extracellular amino acids, supporting intracellular localization of the putative amino acid sensor. In contrast to lower eukaryotes such as yeast, which possess an extracellular amino acid sensor, our findings provide the first direct evidence for an intracellular location for the putative amino acid sensor in animal cells that signals increased amino acid availability to TOR/p70(S6K).     

Drosophila development: novel signal elicits visceral response

A Drosophila screen aimed at furthering understanding of how tissues develop from the mesoderm has identified a novel signalling molecule that is proposed to signal from somatic muscle progenitors to direct the development of adjacent visceral muscle.     

Interchange of DNA-binding modes in the deformed and ultrabithorax homeodomains: a structural role for the N-terminal arm

The deformed (Dfd) and ultrabithorax (Ubx) homeoproteins regulate developmental gene expression in Drosophila melanogaster by binding to specific DNA sequences within its genome. DNA binding is largely accomplished via a highly conserved helix-turn-helix DNA-binding domain that is known as a homeodomain (HD). Despite nearly identical DNA recognition helices and similar target DNA sequence preferences, the in vivo functions of the two proteins are quite different. We have previously revealed differences between the two HDs in their interactions with DNA. In an effort to define the individual roles of the HD N-terminal arm and recognition helix in sequence-specific binding, we have characterized the structural details of two Dfd/Ubx chimeric HDs in complex with both the Dfd and Ubx-optimal-binding site sequences. We utilized hydroxyl radical cleavage of DNA to assess the positioning of the proteins on the binding sites. The effects of missing nucleosides and purine methylation on HD binding were also analyzed. Our results show that both the Dfd and Ubx HDs have similar DNA-binding modes when in complex with the Ubx-optimal site. There are subtle but reproducible differences in these modes that are completely interchanged when the Dfd N-terminal arm is replaced with the corresponding region of the Ubx HD. In contrast, we showed previously that the Dfd-optimal site sequence elicits a very different binding mode for the Ubx HD, while the Dfd HD maintains a mode similar to that elicited by the Ubx-optimal site. Our current methylation interference studies suggest that this alternate binding mode involves interaction of the Ubx N-terminal arm with the minor groove on the opposite face of DNA relative to the major groove that is occupied by the recognition helix. As judged by hydroxyl radical footprinting and the missing nucleoside experiment, it appears that interaction of the Ubx recognition helix with the DNA major groove is reduced. Replacing the Dfd N-terminal arm with that of Ubx does not elicit a complete interchange of the DNA-binding mode. Although the position of the chimera relative to DNA, as judged by hydroxyl radical footprinting, is similar to that of the Dfd HD, the missing nucleoside and methylation interference patterns resemble those of the Ubx HD. Repositioning of amino acid side-chains without wholesale structural alteration in the polypeptide appears to occur as a function of N-terminal arm identity and DNA-binding site sequence. Complete interchange of binding modes was achieved only by replacement of the Dfd N-terminal arm and the recognition helix plus 13 carboxyl-terminal residues with the corresponding residues of Ubx. The position of the N-terminal arm in the DNA minor groove appears to differ in a manner that depends on the two base-pair differences between the Dfd and Ubx-optimal-binding sites. Thus, N-terminal arm position dictates the binding mode and the interaction of the recognition helix with nucleosides in the major groove.     

Crystal structure of a thermostable lipase from Bacillus stearothermophilus P1

We describe the first lipase structure from a thermophilic organism. It shares less than 20% amino acid sequence identity with other lipases for which there are crystal structures, and shows significant insertions compared with the typical alpha/beta hydrolase canonical fold. The structure contains a zinc-binding site which is unique among all lipases with known structures, and which may play a role in enhancing thermal stability. Zinc binding is mediated by two histidine and two aspartic acid residues. These residues are present in comparable positions in the sequences of certain lipases for which there is as yet no crystal structural information, such as those from Staphylococcal species and Arabidopsis thaliana. The structure of Bacillus stearothermophilus P1 lipase provides a template for other thermostable lipases, and offers insight into mechanisms used to enhance thermal stability which may be of commercial value in engineering lipases for industrial uses.     

New structural and mechanistic insight into the A-rule and the instructional and non-instructional behavior of DNA photoproducts and other lesions

The A-rule in mutagenesis was originally proposed to explain the preponderance of X-->T mutations observed for abasic sites and UV damaged sites. It was deduced that when a polymerase was faced with a non-instructional lesion, typified by an abasic site, it would preferentially incorporate an A. In the absence of any other compelling explanation, any lesion causing an X-->T mutation has often been classified as non-instructional to account for its apparent lack of instructional ability. The A-rule and the classification of lesions as non-instructional were formulated before the active sites of any polymerases or the mechanism by which they synthesized DNA were known. Since then, much structural and kinetic data on DNA polymerases has emerged to suggest mechanistic explanations for the A-rule and the instructive and non-instructive behavior of lesions such as cis-syn dimers. Polymerases involved in the replication of undamaged DNA have highly constrained active sites that evolved to only accommodate the templating base and the complementary nucleotide and as a result are relatively intolerant of modifications that alter the size and shape of the nascent base pair. On the other hand, DNA damage bypass polymerases have much more open and less constrained active sites, which are much more tolerant of modifications. An otherwise instructional lesion would become non-instructional if it were unable to fit into the active site, and thereby behave transiently like an abasic site, leading to the insertion of whichever nucleotide is favored by the polymerase, generally an A. In this review, what is known about the active sites and mechanisms of replicative and DNA damage bypass polymerases will be discussed with regard to the A-rule and non-instructive behavior of lesions, typified by dipyrimidine photoproducts.     

Cryptic species in Stachybotrys chartarum

Stachybotrys chartarum has received much attention as a possible cause of sick-building syndrome. Because morphological species recognition in fungi can hide diversity, we applied a phylogenetic approach to search for cryptic species. We examined 23 isolates from the San Francisco Bay Area, and another seven from around the US. Using markers we developed for three polymorphic protein coding loci (chitin synthase 1, beta-tubulin 2, and trichodiene synthase 5), we infer that two distinct phylogenetic species exist within the single described morphological species. We have found no correlation between genetic isolation and geographic distance.     

Isoflavone glycosides from Derris scandens

Five isoflavone glycosides, named derriscandenosides A–E (1–5), were isolated from the stems of Derris scandens, together with ten known compounds comprising one isoflavone, two benzoic acid derivatives, three glucosyl isoflavones and four rhamnosyl-(1→6)-glucosyl isoflavones. The structures of the glycosides were assigned on the basis of spectroscopic data, especially of the acetate derivatives. Three known rhamnosyl-(1→6)-glucosyl isoflavones isolated from a crude fraction were retested for hypotensive activity with varying results.     

Application of non-parametric bootstrap methods to estimate confidence intervals for QTL location in a beef cattle QTL experimental population

Empirical confidence intervals (CIs) for the estimated quantitative trait locus (QTL) location from selective and non-selective non-parametric bootstrap resampling methods were compared for a genome scan involving an Angus x Brahman reciprocal fullsib backcross population. Genetic maps, based on 357 microsatellite markers, were constructed for 29 chromosomes using CRI-MAP V2.4. Twelve growth, carcass composition and beef quality traits (n = 527-602) were analysed to detect QTLs utilizing (composite) interval mapping approaches. CIs were investigated for 28 likelihood ratio test statistic (LRT) profiles for the one QTL per chromosome model. The CIs from the non-selective bootstrap method were largest (87 7 cM average or 79-2% coverage of test chromosomes). The Selective II procedure produced the smallest CI size (42.3 cM average). However, CI sizes from the Selective II procedure were more variable than those produced by the two LOD drop method. CI ranges from the Selective II procedure were also asymmetrical (relative to the most likely QTL position) due to the bias caused by the tendency for the estimated QTL position to be at a marker position in the bootstrap samples and due to monotonicity and asymmetry of the LRT curve in the original sample.     

Subcellular distribution of cadmium and its cellular ligands in mussel digestive gland cells as revealed by combined autometallography and X-ray microprobe analysis

Autometallography (AMG) and electron probe X-ray microanalysis (EPXMA) were applied in combination to determine the subcellular distribution of Cd and its subcellular ligands in the digestive gland cells of Cd-exposed mussels Mytilus galloprovincialis. Black silver deposits (BSD), which reveal the presence of metals when AMG is applied, were only localized in digestive cell lysosomes. Digestive cell cytoplasm and basophilic cells were devoid of BSD. EPXMA (static probe and X-ray mapping) indicated that Cd, S (possibly associated with metallothioneins or metallothionein-like proteins) and autometallographical Ag ions are co-localized within digestive cell lysosomes. In addition, Cd and S co-occur in the absence of Ag in the cytosol of digestive cells. AMG does not reveal the presence of the Cd pool strongly bound to cytosolic Cd-metallothionein complexes; only free Cd or Cd supposedly loosely bound to (semi)digested metallothionein within lysosomes was revealed. The levels of lysosomal Cd were indirectly quantified by stereology as the volume density of BSD (Vv_BSD). Significantly higher values were recorded in Cd-exposed mussels compared with controls at all exposure times. However, Vv_BSD values were lower at days 7 and 21 than at day 1. This relative decrease in Vv_BSD reflected another (and confounding) response elicited by Cd-exposure in the digestive epithelium: the volume density of basophilic cells (Vv_BAS) increased significantly as exposure progressed. Due to this cell-type replacement, the net accumulative capacity of the digestive epithelium decreases at long exposure times.     

Environmental stress causes oxidative damage to plant mitochondria leading to inhibition of glycine decarboxylase

A cytotoxic product of lipid peroxidation, 4-hydroxy-2-nonenal (HNE), rapidly inhibited glycine, malate/pyruvate, and 2-oxoglutarate-dependent O2 consumption by pea leaf mitochondria. Dose- and time-dependence of inhibition showed that glycine oxidation was the most severely affected with a K(0.5) of 30 microm. Several mitochondrial proteins containing lipoic acid moieties differentially lost their reactivity to a lipoic acid antibody following HNE treatment. The most dramatic loss of antigenicity was seen with the 17-kDa glycine decarboxylase complex (GDC) H-protein, which was correlated with the loss of glycine-dependent O2 consumption. Paraquat treatment of pea seedlings induced lipid peroxidation, which resulted in the rapid loss of glycine-dependent respiration and loss of H-protein reactivity with lipoic acid antibodies. Pea plants exposed to chilling and water deficit responded similarly. In contrast, the damage to other lipoic acid-containing mitochondrial enzymes was minor under these conditions. The implication of the acute sensitivity of glycine decarboxylase complex H-protein to lipid peroxidation products is discussed in the context of photorespiration and potential repair mechanisms in plant mitochondria.