A transcriptional response to Wnt protein in human embryonic carcinoma cells

Background  

Wnt signaling is implicated in many developmental decisions, including stem cell control, as well as in cancer. There are relatively few target genes known of the Wnt pathway.     

Leaf carbohydrate responses to CO2 enrichment at the top of a tropical forest

The accumulation of non-structural leaf carbohydrates is one of the most consistent plant responses to elevated CO₂. It has been found in both fast-and slow-growing plants and is largely independent of the duration of exposure. Changes in leaf quality are thus to be expected, irrespective of other plant responses to atmospheric CO₂ enrichment. However, there is no experimental evidence from tropical forests, the biome with the largest biomass carbon pool. Here we report in situ mesophyll responses of mature tropical trees to a doubling of CO₂. Individually CO₂-enriched leaves on 25 to 35-m-tall forest trees living at 26–35°C can be assumed to experience little sink limitation, and so, may be expected to exhibit no or very little carbohydrate accumulation. We tested this hypothesis using the leaf cup method on leaves accessible via the canopy crane of the Smithsonian Tropical Research Institute in a semi-deciduous tropical forest in Panamá. We also investigated the influence of the leaf-specific light regime, another possible environmental determinant of leaf carbon gain and mobile leaf carbohydrates. Total non-structural carbohydrates (TNC) reached a new steady state concentration after less than 4 days of exposure to twice ambient CO₂ concentration. Against expectation, all four tree species investigated (Anacardium excelsum, Cecropia longipes, C. peltata, Ficus insipida) accumulated significant amounts of TNC (+41 to +61%) under elevated CO₂. The effect was stronger at the end of the daylight period (except for Ficus), but was still significant in all four species at the end of the dark period. In contrast, neither artificial nor natural shading affected leaf TNC. Taken together, these observations suggest that TNC accumulation reflects a mesophyll-bound tissue response specific to elevated CO₂, presumably unrelated to sink limitations. Thus, leaves of tropical forests seem not to be an exception, and will most likely contain more non-structural carbohydrates in a CO₂-rich world. Received: 28 January 1998 / Accepted: 9 April 1998     

Messages from ultrahigh resolution crystal structures

The ever growing availability of macromolecular crystal structures determined at atomic resolution has now reached a critical size, making it possible to obtain statistically unbiased data on both protein stereochemistry and the validity of the parameters used in their refinement. Besides the determination of the precise geometry of proteins and their active sites, high resolution structures have made it possible to check the application of normal mode calculations, to calculate charge density distributions and to analyze hydration shells around protein molecules. Even if only a few structures involve protein complexes, either with ligands or prosthetic groups, the information obtained in these cases is of great interest for obtaining the physical parameters of these interactions.     

Contact sex signals on web and cuticle of Tegenaria atrica (Araneae,Agelenidae)

Qualitative changes in lipid content and composition were examined on the web and cuticle of Tegenaria atrica females in relation to sexual receptivity. In this spider species, 78 different compounds were detected by gas chromatography in the cuticle extract and 50 in the web; 28 identical compounds were present both on the spider silk and the cuticle of the female. The components were long‐chain aliphatic hydrocarbons, fatty acids, and esters. On the web, sexual receptivity was correlated with changes in eleven polar compounds. On the cuticle, sexual receptivity was correlated with changes in eight polar compounds and 26 hydrocarbons. Bioassays demonstrated that the methanol eluate of webs and females were involved in stimulating the sexual behavior of males. Qualitative and/or quantitative changes in hexadecanoic acid, octadecadienoic acid, octadecenoic acid, methyl palmitate, methyl octadecanoate, and n‐tricosane could play a role in the contact sex signals from web and cuticle of T. atrica. Arch. Insect Biochem. Physiol. 40:194–202, 1999. © 1999 Wiley‐Liss, Inc.     

Impact of peptides on the recognition of HLA class I molecules by human HLA antibodies

MHC class I molecules expressed on cell surfaces are composed of H chain, beta2-microglobulin and any of a vast array of peptides. The role of peptide in the recognition of HLA class I by serum HLA Abs is unknown. In this study, the solid-phase assay of a series (n = 11) of HLA-A2-reactive, pregnancy-induced, human mAbs on a panel (n = 12) of recombinant monomeric HLA-A2 molecules, each containing a single peptide, revealed peptide selectivity of the mAbs. The flow cytometry membrane staining intensities on the HLA-A2-transduced cell line K562, caused by these mAbs, correlated with the number of monomer species detected by the mAbs. Flow cytometry staining on HLA-A2-bearing cell lines of a variety of lineages was indicative of tissue selectivity of these HLA-A2 mAbs. This tissue selectivity suggests that the deleterious effect on allografts is confined to alloantibodies recognizing only HLA class I loaded with peptides that are derived from tissue-specific and household proteins. Since Abs that are only reactive with HLA loaded with irrelevant peptides are expected to be harmless toward allografts, the practice of HLA Ab determination on lymphocyte-derived HLA deserves reconsideration.     

Enzyme-substrate complex structures of a GH39 beta-xylosidase from Geobacillus stearothermophilus

Beta-D-Xylosidases are glycoside hydrolases that catalyse the release of xylose units from short xylooligosaccharides and are engaged in the final breakdown of plant cell-wall hemicelluloses. beta-D-Xylosidases are found in glycoside hydrolase families 3, 39, 43, 52 and 54. The first crystal structure of a GH39 beta-xylosidase revealed a multi-domain organization with the catalytic domain having the canonical (beta/alpha)8 barrel fold. Here, we report the crystal structure of the GH39 Geobacillus stearothermophilus beta-D-xylosidase, inactivated by a point mutation of the general acid-base residue E160A, in complex with the chromogenic substrate molecule 2,5-dinitrophenyl-beta-D-xyloside. Surprisingly, six of the eight active sites present in the crystallographic asymmetric unit contain the trapped covalent glycosyl-enzyme intermediate, while two of them still contain the uncleaved substrate. The structural characterization of these two critical species along the reaction coordinate of this enzyme identifies the residues forming its xyloside-binding pocket as well as those essential for its aglycone recognition.     

Optimisation of insect cell growth in deep-well blocks: development of a high-throughput insect cell expression screen

This report describes a method to culture insects cells in 24 deep-well blocks for the routine small-scale optimisation of baculovirus-mediated protein expression experiments. Miniaturisation of this process provides the necessary reduction in terms of resource allocation, reagents, and labour to allow extensive and rapid optimisation of expression conditions, with the concomitant reduction in lead-time before commencement of large-scale bioreactor experiments. This therefore greatly simplifies the optimisation process and allows the use of liquid handling robotics in much of the initial optimisation stages of the process, thereby greatly increasing the throughput of the laboratory. We present several examples of the use of deep-well block expression studies in the optimisation of therapeutically relevant protein targets. We also discuss how the enhanced throughput offered by this approach can be adapted to robotic handling systems and the implications this has on the capacity to conduct multi-parallel protein expression studies.     

Duplication and transposition of the<Emphasis Type="Italic"> NF1</Emphasis> pseudogene regions on chromosomes 2, 14, and 22

Numerous NF1 pseudogenes have been identified in the human genome. Those in 2q21, 14q11, and 22q11 form a subset with a similar genomic organization and a high sequence homology. We have studied, by polymerase chain reaction and fluorescence in situ hybridization, the extent of homology of the regions surrounding these NF1 pseudogenes. Our analyses have demonstrated that a fragment of at least 640 kb is homologous between the three regions. Based on previous studies and these new findings, we propose a model for the spreading of the NF1 pseudogene-containing regions. A fragment of approximately 640 kb was first duplicated in chromosome region 2q21 and transposed to 14q11. Subsequently, this fragment was duplicated in 14q11 and transposed to 22q11. A part of the 640-kb fragment in 14q11, with a length of about 430 kb, was further duplicated to a variable extent in 14q11. In addition, we have identified sequences that may facilitate the duplication and transposition of the 640-kb and 430-kb fragments.