Construction of two ordered cDNA libraries enriched in genes encoding plasmalemma and tonoplast proteins from a high-efficiency expression library

We constructed a high-efficiency expression library from Arabidopsis cDNA clones by introducing a poly (dC) stretch at the 5' end of the clones. This library enables the synthesis of proteins from all the cDNA clones present. We have screened the high-efficiency expression library with antibodies raised against total proteins from Arabidopsis plasmalemma and tonoplast. With the positive clones, we have constructed two cDNA ordered libraries enriched in genes encoding plasmalemma (522 clones) and tonoplast proteins (594 clones). Partial sequencing of both libraries shows that a high proportion (47%) of the clones encoded putative membrane proteins, or membrane-associated proteins. When sequenced, 55% of the cDNAs were new EST sequences for Arabidopsis, 26% were similar to genes present in other plants or organisms, and 29% were not referenced in any databank. Immunoscreening of the two cDNA ordered libraries with antibodies raised against proteins from Arabidopsis cells submitted to osmotic stress allows the selection of genes over- and under-expressed in stress conditions.     

Molecular Dynamics in Protein—Single Stranded DNA Complexes. Two Distinct Nucleoside Mobilities in Poly(deoxythymidylic acid)—poly-L-lysine Complexes

Abstract

Stoichiometric amounts of poly-L-lysine were added to site-specifically spin labeled single stranded nucleic acids and the resulting complexes analyzed by electron spin resonance spectroscopy (ESR). The nucleic acids were spin labeled to different extents and with labels of varying tether length. The ESR data are used to determine nucleoside dynamics and some structural features in these complexes. It is concluded that two distinct base mobilities exist in the complexes; one set is characterized by a mean correlation time τR = 2 ns, and the other one by a τR ≤ 50 ns. A model is proposed which suggests that a poly-L-lys single stranded nucleic acid complex consists of low mobility segments flanked by more mobile bases. An interesting feature of the proposed model is its applicability to explain ESR data of single strand binding protein-spin labeled nucleic acid complexes, which can also be interpreted in terms of two distinct nucleoside mobility states. It is hypothesized that this phenomenon could be of biological significance for the release of protein ligands from a protein-nucleic acid complex.     

Über die direkte und indirekte Strahlenwirkung auf Enzyme in der Zelle

Zusammenfassung Es wurde die Röntgenstrahleninaktivierung von Laktatdehydrogenase in Rattenleber und Invertase in Hefe untersucht. Die Inaktivierung war in den feuchten lebenden Zellen und in den getrockneten Zellen praktisch gleich, obwohl ein Beitrag der indirekten Strahlenwirkung in den feuchten Zellen zu erwarten war.Auszugsweise vorgetragen auf dem XI. Internationalen Kongreß für Radiologie, Rom, September 1965     

Semi‐parametric analysis of overdispersed count and metric data with varying follow‐up times: Asymptotic theory and small sample approximations

Count data are common endpoints in clinical trials, for example magnetic resonance imaging lesion counts in multiple sclerosis. They often exhibit high levels of overdispersion, that is variances are larger than the means. Inference is regularly based on negative binomial regression along with maximum‐likelihood estimators. Although this approach can account for heterogeneity it postulates a common overdispersion parameter across groups. Such parametric assumptions are usually difficult to verify, especially in small trials. Therefore, novel procedures that are based on asymptotic results for newly developed rate and variance estimators are proposed in a general framework. Moreover, in case of small samples the procedures are carried out using permutation techniques. Here, the usual assumption of exchangeability under the null hypothesis is not met due to varying follow‐up times and unequal overdispersion parameters. This problem is solved by the use of studentized permutations leading to valid inference methods for situations with (i) varying follow‐up times, (ii) different overdispersion parameters, and (iii) small sample sizes.     

Specificity determinants of human cathepsin s revealed by crystal structures of complexes

Cathepsin S, a lysosomal cysteine protease of the papain superfamily, has been implicated in the preparation of MHC class II alphabeta-heterodimers for antigen presentation to CD4+ T lymphocytes and is considered a potential target for autoimmune-disease therapy. Selective inhibition of this enzyme may be therapeutically useful for attenuating the hyperimmune responses in a number of disorders. We determined the three-dimensional crystal structures of human cathepsin S in complex with potent covalent inhibitors, the aldehyde inhibitor 4-morpholinecarbonyl-Phe-(S-benzyl)Cys-Psi(CH=O), and the vinyl sulfone irreversible inhibitor 4-morpholinecarbonyl-Leu-Hph-Psi(CH=CH-SO(2)-phenyl) at resolutions of 1.8 and 2.0 A, respectively. In the structure of the cathepsin S-aldehyde complex, the tetrahedral thiohemiacetal adduct favors the S-configuration, in which the oxygen atom interacts with the imidazole group of the active site His164 rather than with the oxyanion hole. The present structures provide a detailed map of noncovalent intermolecular interactions established in the substrate-binding subsites S3 to S1' of cathepsin S. In the S2 pocket, which is the binding affinity hot spot of cathepsin S, the Phe211 side chain can assume two stable conformations that accommodate either the P2-Leu or a bulkier P2-Phe side chain. This structural plasticity of the S2 pocket in cathepsin S explains the selective inhibition of cathepsin S over cathepsin K afforded by inhibitors with the P2-Phe side chain. Comparison with the structures of cathepsins K, V, and L allows delineation of local intermolecular contacts that are unique to cathepsin S.     

Solubilization of galactosyltransferase that synthesizes 1,4-beta-galactan side chains in pectic rhamnogalacturonan I

β -1,4-Galactan galactosyltransferase (GT) activity was solubilized from potato microsomal membranes in the presence of 78 m M 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonic acid. The solubilized GT activity transferred ¹⁴[C]galactose from UDP-¹⁴[C]galactose onto the acceptor-substrates composed of rhamnogalacturonan (RG) with short galactan chains (RG-A, approximately 1.2 MDa, mol% Gal/Rha = 0.7; RG-B, approximately 21 kDa, mol% Gal/Rha = 1.2). However, shorter RG containing short galactan chains (approximately 2 kDa and 1.2 kDa), RG oligomers without galactosyl-residues, galactan, and galactooligomers did not act as acceptor-substrates. Optimal pH for ¹⁴[C] incorporation onto RG-A and RG-B was around 5.6 and 7.5, respectively. The ¹⁴[C]-labelled products synthesized upon RG-A and RG-B could be digested with a RG specific lyase into smaller RG fragments. 1,4- β - Endog alactanase could not digest the former product, whereas the latter product was digested to ¹⁴[C]galactobiose and ¹⁴[C]galactose. This demonstrates that at least two GT activities were solubilized from potato microsomal membranes. One had optimal pH around 5.6 to transfer galactosyl residues onto RG-A, whereas the other had optimal pH around 7.5 to transfer galactosyl residues onto RG-B. Both synthesized galactan attached to the RG backbone of RG-A and RG-B, and the galactan synthesized onto the RG-B acceptor was 1,4- β -linked.     

AXY8 encodes an α-fucosidase, underscoring the importance of apoplastic metabolism on the fine structure of Arabidopsis cell wall polysaccharides

An Arabidopsis thaliana mutant with an altered structure of its hemicellulose xyloglucan (XyG; axy-8) identified by a forward genetic screen facilitating oligosaccharide mass profiling was characterized. axy8 exhibits increased XyG fucosylation and the occurrence of XyG fragments not present in the wild-type plant. AXY8 was identified to encode an α-fucosidase acting on XyG that was previously designated FUC95A. Green fluorescent protein fusion localization studies and analysis of nascent XyG in microsomal preparations demonstrated that this glycosylhydrolase acts mainly on XyG in the apoplast. Detailed structural analysis of XyG in axy8 gave unique insights into the role of the fucosidase in XyG metabolism in vivo. The genetic evidence indicates that the activity of glycosylhydrolases in the apoplast plays a major role in generating the heterogeneity of XyG side chains in the wall. Furthermore, without the dominant apoplastic glycosylhydrolases, the XyG structure in the wall is mainly composed of XXXG and XXFG subunits.     

CASANOVA: Permutation inference in factorial survival designs

We propose inference procedures for general factorial designs with time-to-event endpoints. Similar to additive Aalen models, null hypotheses are formulated in terms of cumulative hazards. Deviations are measured in terms of quadratic forms in Nelson–Aalen-type integrals. Different from existing approaches, this allows to work without restrictive model assumptions as proportional hazards. In particular, crossing survival or hazard curves can be detected without a significant loss of power. For a distribution-free application of the method, a permutation strategy is suggested. The resulting procedures' asymptotic validity is proven and small sample performances are analyzed in extensive simulations. The analysis of a data set on asthma illustrates the applicability.     

Respect for outsiders? respect for the law? the moral evaluation of high scale issues by US immigration officers

High-scale morality is the study of moral ideas and sentiments deployed in relations that encompass multiple, geographically or socially distant populaces. The envisioning of distant people, their attributed moral personhood, the evaluation of their perceived behaviour, and the rectification of wrongs through the use of powerful organizations are key topics in high-scale morality. High-scale morality differs from existing anthropological approaches that emphasize local ethnography or contrastive moral ideas; it addresses the moralization of issues like world hunger, the drug trade, or international migration. The officers of the US Immigration and Naturalization Service understand and evaluate legal and illegal immigrants, as well as directly enacting moral rectification for the US polity. As they resolve moral dilemmas on their job, they utilize pervasive models for moral thought and action in capitalist, individualist, stratified, and bureaucratized societies. The article finishes by considering directions in which anthropology can contribute to understanding the moral dimension of global issues.