Crystal structure of the alkaline proteinase Savinase from Bacillus lentus at 1.4 A resolution.

Savinase (EC3.4.21.14) is secreted by the alkalophilic bacterium Bacillus lentus and is a representative of that subgroup of subtilisin enzymes with maximum stability in the pH range 7 to 10 and high activity in the range 8 to 12. It is therefore of major industrial importance for use in detergents. The crystal structure of the native form of Savinase has been refined using X-ray diffraction data to 1.4 A resolution. The starting model was that of subtilisin Carlsberg. A comparison to the structures of the closely related subtilisins Carlsberg and BPN' and to the more distant thermitase and proteinase K is presented. The structure of Savinase is very similar to those of homologous Bacillus subtilisins. There are two calcium ions in the structure, equivalent to the strong and the weak calcium-binding sites in subtilisin Carlsberg and subtilisin BPN', well known for their stabilizing effect on the subtilisins. The structure of Savinase shows novel features that can be related to its stability and activity. The relatively high number of salt bridges in Savinase is likely to contribute to its high thermal stability. The non-conservative substitutions and deletions in the hydrophobic binding pocket S1 result in the most significant structural differences from the other subtilisins. The different composition of the S1 binding loop as well as the more hydrophobic character of the substrate-binding region probably contribute to the alkaline activity profile of the enzyme. The model of Savinase contains 1880 protein atoms, 159 water molecules and two calcium ions. The crystallographic R-factor [formula; see text].     

The reality of anthropology

The article concerns the continuity between anthropology and the reality it studies. Discovery and definition merge. This is substantiated by a discussion of ‘events’ and ‘worlds’. While the anthropologist becomes her own informant in the field, in the sense that her experience informs her ethnography, anthropology simultaneously shapes her discoveries. This simultaneity ultimately dissolves the distinction between subject and object, and from this point anthropology may recondition the conditions of science.     

Unsaturated Free Fatty Acids Increase Benzodiazepine Receptor Agonist Binding Depending on the Subunit Composition of the GABAA Receptor Complex

Abstract: It has been shown previously that unsaturated free fatty acids (FFAs) strongly enhance the binding of agonist benzodiazepine receptor ligands and GABA_A receptor ligands in the CNS in vitro. To investigate the selectivity of this effect, recombinant human GABA_A/benzodiazepine receptor complexes formed by different subunit compositions (α_xβ_yγ₂, x = 1, 2, 3, and 5; y = 1, 2, and 3) were expressed using the baculovirus-transfected Sf9 insect cell system. At 10^?4M, unsaturated FFAs, particularly arachidonic (20:4) and docosahexaenoic (22:6) acids, strongly stimulated (>200% of control values) the binding of [³H]flunitrazepam ([³H]FNM) to the α₃β₂γ₂ receptor combination in whole cell preparations. No effect or small increases in levels of unsaturated FFAs on [³H]FNM binding to α₁β_xγ₂ and α₂β_xγ₂ receptor combinations were observed, and weak effects (130% of control values) were detected using the α₅β₂γ₂ receptor combination. The saturated FFAs, stearic and palmitic acids, were without effect on [³H]FNM binding to any combination of receptor complexes. The hydroxylated unsaturated FFAs, ricinoleic and ricinelaidic acids, were shown to decrease the binding of [³H]FNM only if an α₁β₂γ₂ receptor combination was used. Given the heterogeneity of the GABA_A/benzodiazepine receptor subunit distribution in the CNS, the effects of FFAs on the benzodiazepine receptor can be assumed to vary at both cellular and regional levels.     

Glycine transporter dimers: evidence for occurrence in the plasma membrane

Different Na(+)/Cl(-)-dependent neurotransmitter transporters of the SLC6a family have been shown to form dimers or oligomers in both intracellular compartments and at the cell surface. In contrast, the glycine transporters (GlyTs) GlyT1 and -2 have been reported to exist as monomers in the plasma membrane based on hydrodynamic and native gel electrophoretic studies. Here, we used cysteine substitution and oxidative cross-linking to show that of GlyT1 and GlyT2 also form dimeric complexes within the plasma membrane. GlyT oligomerization at the cell surface was confirmed for both GlyT1 and GlyT2 by fluorescence resonance energy transfer microscopy. Endoglycosidase treatment and surface biotinylation further revealed that complex-glycosylated GlyTs form dimers located at the cell surface. Furthermore, substitution of tryptophan 469 of GlyT2 by an arginine generated a transporter deficient in dimerization that was retained intracellulary. Based on these results and GlyT structures modeled by using the crystal structure of the bacterial homolog LeuT(Aa), as a template, residues located within the extracellular loop 3 and at the beginning of transmembrane domain 6 are proposed to contribute to the dimerization interface of GlyTs.     

Closing Ranks: Fundamentals in History,Politics and Anthropology

In this presentation, I discuss fundamentalism from a processual perspective, seeking to tease out some general qualities of the processes involved in a return to fundamentals amidst social change. I start with an analysis of the historical dynamics of Icelandic society in the period 1400–1800, showing how the increasing insistence on old patterns and cultural fundamentals contributed to the gradual destruction of a one time flourishing medieval society. This devolution, I suggest, is closely correlated with a process of amplification (Sahlins) of a particular set of values, leading to a loss of flexibility in the response to environmental and other changes. Next follows a discussion of present day concerns with nationalism and other interests in bounding oneself off from the surrounding world, and demanding recognition in return. One of the processes discussed is a process of transvaluation (Tambiah), assimilating particulars to a larger and less context‐bound scheme and thereby gradually deepening the cleavage between selves and others, sometimes to the point of epistemological closure (Ignatieff). Finally, one of the anthropological fundamentals, holism, is discussed with a view to reassessing its potential for present‐day anthropology. It is argued that through the process of knowing implied in fieldwork, anthropologists arrive at a dual understanding of perceived wholes and creative agents. A new sense of holism may still grant both consistency and uniqueness to the anthropological discipline.     

Introduction of a free cysteinyl residue at position 68 in the subtilisin Savinase, based on homology with proteinase K.

Two subfamilies of the subtilisins, distinguished by the presence or absence of a free cysteinyl residue near the essential histidyl residue of the catalytic triad, are known. In order to evaluate the significance of the presence of this -SH group a cysteinyl residue has been introduced by site-directed mutagenesis into the cysteine-free subtilisin-like enzyme from Bacillus lentus, i.e. Savinase. The free cysteine affects the enzyme activity only slightly but renders it sensitive to mercurials presumably due to an indirect effect. The results indicate that the -SH group is not involved in catalysis.     

MOLECULAR PHYLOGENY OF SELECTED SPECIES OF THE ORDER DINOPHYSIALES (DINOPHYCEAE)—TESTING THE HYPOTHESIS OF A DINOPHYSIOID RADIATION1

Almost 80 years ago, a radiation scheme based on structural resemblance was first outlined for the marine order Dinophysiales. This hypothetical radiation illustrated the relationship between the dinophysioid genera and included several independent, extant lineages. Subsequent studies have supplied additional information on morphology and ecology to these evolutionary lineages. We have for the first time combined morphological information with molecular phylogenies to test the dinophysioid radiation hypothesis in a modern context. Nuclear‐encoded LSU rDNA sequences including domains D1‐D6 from 27 species belonging to Dinophysis Ehrenb., Ornithocercus F. Stein, Phalacroma F. Stein, Amphisolenia F. Stein, Citharistes F. Stein, and Histioneis F. Stein were obtained from the Indian Ocean. Previously, LSU rDNA has only been determined from one of these. In Bayesian analyses, Amphisolenia formed a long basal clade to the other dinophysioids. These diverged into two separate lineages, the first comprised species with a classical Phalacroma outline, also including the type species P. porodictyum F. Stein. Thus, we propose to reinstate the genus Phalacroma. The relationship between the genera in the second lineage was not well resolved. However, the molecular phylogeny supported monophyly of Histioneis and Citharistes and showed the genus Dinophysis to be polyphyletic and in need of a taxonomic revision. Species of Ornithocercus grouped with Citharistes, but this relationship remained unresolved. The phylogenetic trees furthermore revealed convergent evolution of several morphological characters in the dinophysioids. According to the molecular data, the dinophysioids appeared to have evolved quite differently from the radiation schemes previously hypothesized. Four dinophysioid species had identical LSU rDNA sequences to other well‐established species.     

Camillea (Xylariaceae, Ascomycota), including two new species, along a trans-Andean altitude gradient in Ecuador

Two new species of Camillea are described from localities on an east–west transect through Ecuador between the latitudes 1°00’N and 1°00’S. Camillea ovalispora was collected in San Vicente de Huaticocha, an area characterized as humid lowland rainforest and premontane cloud forest; C. unistoma was collected at Cuyabeno, a protected area with black water inundated forest and primary rainforest on terra firme. Further, C. heterostoma var. macrospora is elevated to full species status as C. macrospora. New country records of six Camillea species are annotated from the same east-west transect. These are C. flosculosa, C. fossulata, C. hainesii, C. macrospora, C. scriblita, and C. tinctor. Also, previously recorded species have been annotated with new specimens added. Additionally, six distinct taxa from the transect that appear to be undescribed were included in the analysis; one is partly described but not the others due to insufficient material. The data showed an obvious altitude influence on the distribution and frequency of Camillea species. The diversity of species is lower on the western part of the transect compared to the eastern side, probably due to multiple factors such as differences in search intensity between the two slopes, difference in annual rainfall, and possibly the availability of host plants. Camillea taxa were only found at altitudes below 2,000 m, with the highest diversity below 600 m. Palmae is reported as a new host for Camillea species.