Structure of astacin and implications for activation of astacins and zinc-ligation of collagenases.

Astacin, a digestive zinc-endopeptidase from the crayfish Astacus astacus L., is the prototype for the 'astacin family', which includes mammalian metallo-endopeptidases and developmentally regulated proteins of man, fruitfly, frog and sea urchin. Here we report the X-ray crystal structure of astacin, which reveals a deep active-site cleft, with the zinc at its bottom ligated by three histidines, a water molecule and a more remote tyrosine. The third histidine (His 102) forms part of a consensus sequence, shared not only by the members of the astacin family, but also by otherwise sequentially unrelated proteinases, such as vertebrate collagenases. It may therefore represent the elusive 'third' zinc ligand in these enzymes. The amino terminus of astacin is buried forming an internal salt-bridge with Glu 103, adjacent to His 102. Astacin pro-forms extended at the N terminus, as observed for some 'latent' mammalian astacin homologues, did not exhibit this 'active' conformation, indicating an activation mechanism reminiscent of trypsin-like serine proteinases.     

Development and function of T cells in mice rendered interleukin-2 deficient by gene targeting.

Interleukin-2 (IL-2) is a lymphocytotropic hormone which is thought to have a key role in the immune response of mammalian cells. It is produced by a subpopulation of activated T-lymphocytes and acts in vitro as the principal auto- and paracrine T-cell growth factor (for reviews see refs 1-3). IL-2 is, however, not the sole T-cell growth factor, nor does it act exclusively on T cells, also promoting growth of NK cells and differentiation of B cells. A role for IL-2 in T-cell development has been postulated but remains controversial. Here we test the requirement for IL-2 in vivo using IL-2-deficient mice generated by targeted recombination. We find that mice homozygous for the IL-2 gene mutation are normal with regard to thymocyte and peripheral T-cell subset composition, but that a dysregulation of the immune system is manifested by reduced polyclonal in vitro T-cell responses and by dramatic changes in the isotype levels of serum immunoglobulins.     

A small GTP-binding protein dissociates from synaptic vesicles during exocytosis.

Low-molecular-weight GTP-binding proteins are strong candidates for regulators of membrane traffic. In yeast, mutations in the sec4 or ypt1 genes encoding small GTP-binding proteins inhibit constitutive membrane flow at the plasma membrane or Golgi complex, respectively. It has been suggested that membrane fusion-fission events are regulated by cycling of small GTP-binding proteins between a membrane-bound and free state, but although most of these small proteins are found in both soluble and tightly membrane-bound forms, there is no direct evidence to support such cycling. In rat brain a small GTP-binding protein, rab3A, is exclusively associated with synaptic vesicles, the secretory organelles of nerve terminals. Here we use isolated nerve terminals to study the fate of rab3A during synaptic vesicle exocytosis. We find that rab3A dissociates quantitatively from the vesicle membrane after Ca2(+)-dependent exocytosis and that this dissociation is partially reversible during recovery after stimulation. These results are direct evidence for an association-dissociation cycle of a small GTP-binding protein during traffic of its host membrane.     

The European Science Foundation: Excerpts from the annual report for 1987

Science Policy News

The European Science Foundation: Excerpts from the annual report for 1987     

Pharmacodynamic profile of CQP 201-403, a novel 8α-amino-ergoline

Summary The profile of action in animals of CQP 201-403, a novel 8-amino-ergoline, is in most aspects that of a very potent dopaminomimetic, both as a prolactin secretion inhibitor, and at the levels of the CNS and the cardiovascular system. Qualitatively CQP 201-403 differs slightly from bromocriptine and apomorphine in its effects on the CNS (no influence on serotonin metabolism in the rat cortex; induction of masculine mounting behavior in rats) and the cardiovascular system of the dog (reflex tachycardia in response to a blood-pressure fall). In man the new compound proved to be highly active in lowering prolactin serum levels and to be more potent than bromocriptine (Parlodel^®).In memory of Dr Annemarie Closse, who died 14 June 1987.     

Free R value: a novel statistical quantity for assessing the accuracy of crystal structures

The determination of macromolecular structure by crystallography involves fitting atomic models to the observed diffraction data. The traditional measure of the quality of this fit, and presumably the accuracy of the model, is the R value. Despite stereochemical restraints, it is possible to overfit or 'misfit' the diffraction data: an incorrect model can be refined to fairly good R values as several recent examples have shown. Here I propose a reliable and unbiased indicator of the accuracy of such models. By analogy with the cross-validation method of testing statistical models I define a statistical quantity (R(free) (T) that measures the agreement between observed and computed structure factor amplitudes for a 'test' set of reflections that is omitted in the modelling and refinement process. As examples show, there is a high correlation between R(free) (T) and the accuracy of the atomic model phases. This is useful because experimental phase information is usually inaccurate, incomplete or unavailable. I expect that R(free) (T) will provide a measure of the information content of recently proposed models of thermal motion and disorder, time-averaging and bulk solvent.     

Purification of platelet-derived endothelial cell growth inhibitor and its characterization as transforming growth factor-β type 1

In 1986, Brown and Clemmons (Proc. natl Acad. Sci. USA83 (1986) 3321) showed that platelets contain a substance, platelet-derived growth inhibitor (PDGI), that inhibits in vitro endothelial cell replication. Although platelets are rich in transforming grwoth factor (TGF-), PDGI was considered not to be related to TGF-, on the basis of its reported properties (extraction from platelets at neutral pH, binding to heparin-Sepharose). However, we purified PDGI to near homogeneity and showed that on the basis of HPLC retention behavior, in vitro growth inhibitory activities with several cell types, receptor binding, and immunoneutralization of growth inhibitory activity with specific anti-TGF- type 1 antibodies, PDGI is most probably identical with TGF- type 1.     

<Emphasis Type="Italic">Naja siamensis</Emphasis>, a cryptic species of venomous snake revealed by mtDNA sequencing

Because of possible variation in venom composition, an understanding of venomous snake systematics is of great importance for the optimization of antivenom treatment of snakebite patients. Intraspecific variation in the morphology of many venomous snakes complicates the definition and indentification of some species when allopatric populations are involved. Selectively neutral or near-neutral mtDNA sequences can reveal evolutionary relationships obscured by ecogenetically-caused morphological variation. We use comparative sequencing of the cytochrome oxidase subunit 1 gene to reveal the existence of a widespread, cryptic species of spiting cobra from southeast Asia. This species,Naja siamensis, is widely sympatric with other Asiatic cobra species. This may be of considerable medical significance, and calls for further research into venom composition in Asiatic cobras.