p36, the major cytoplasmic substrate of src tyrosine protein kinase, binds to its p11 regulatory subunit via a short amino-terminal amphiphatic helix.

Protein I is a hetero-tetramer which contains two copies each of p11 and p36. p36 (calpactin I, lipocortin II) is a major substrate of retrovirally encoded tyrosine protein kinases, while p11 modulates several Ca2+-induced properties also displayed by p36 alone. Here we have characterized the p11 binding site on p36 by fluorescence spectroscopy using porcine p36 labelled at cysteine 8 with the fluorophore Prodan (6-proprionyl-2-dimethylamino-naphthalene). We have used peptides of differing length from the amino-terminal domain of p36 to restrict the major binding site to the first 12 residues. Noticeable binding is still observed with a peptide containing only the first nine residues. Interestingly the N-terminal acetyl group of p36 forms a functional part of the p11 binding site. CD studies indicate that the binding region can form an alpha-helix, which seems to have amphiphatic properties when projected on a helical wheel. This structural element is also known for a calmodulin binding protein. Thus the question is raised whether other p11/calmodulin-related proteins interact with their target proteins via a similar mechanism. We also discuss how p11 could modulate p36 associated properties.     

Water-maze learning and effects of cholinergic drugs in mouse strains with high and low hippocampal pyramidal cell counts

Morphological differences have been found in inbred strains of mice in the number and volume of pyramidal cells in Ammon's horn of the hippocampus. Among the mouse strains surveyed, NZB/BINJ (NZB) and C57BL/10J (B10) are most divergent in both total volume and total number of neurons. These genetically derived differences were exploited to determine hippocampal involvement in the acquisition of a spatial water maze. Genetic differences in hippocampal cell number were related to the acquisition of this spatial task. Mice with small numbers of hippocampal pyramidal cells, the B10 strain, acquired a water-maze task more slowly than either NZB mice or (NZBxNZW) F1 (NZBWF) animals. In addition, strain differences in responsivity to cholinergic manipulations were found. B10 mice were more sensitive than NZB or NZBWF mice to both the disruptive effects of scopolamine and the facilitory effects of physostigmine on swim maze learning. Although other inherited differences undoubtedly exist between these strains as is apparent in other mouse lines, these data suggest a prominent role for the hippocampus in the learning of spatially oriented behavior. Furthermore, this behavior appears to be responsive to cholinergic manipulations.     

Biotransformation of β-myrcene to geraniol by a strain of Rhodococcus erythropolis isolated by selective enrichment from hop plants

The biocatalytic generation of high-value chemicals from abundant, cheap and renewable feedstocks is an area of great contemporary interest. A strain of Rhodococcus erythropolis designated MLT1 was isolated by selective enrichment from the soil surrounding hop plants, using the abundant triene β-myrcene from hops as a sole carbon source for growth. Resting cells of the organism were challenged with β-myrcene, and the major product of biotransformation was determined by mass spectrometric analysis to be the monoterpene alcohol geraniol. Controls demonstrated that the product was biogenic and that an aerobic environment was required. The ability to transform β-myrcene was shown to be restricted to cells that had been grown on this substrate as sole carbon source. Pre-incubation of cells with the cytochrome P450 inhibitors metyrapone or 1-aminobenzotriazole reduced geraniol production by 23% and 73% respectively, but reduction in activity was found not to correlate with the inhibitor concentration. A comparative analysis of insoluble and soluble cell extracts derived from cells of MLT1 grown on either β-myrcene or glucose revealed at least four proteins that were clearly overproduced in response to growth on β-myrcene. Mass spectrometric analysis of tryptic digests of three of these protein bands suggested their identities as an aldehyde dehydrogenase, an acyl-CoA dehydrogenase and a chaperone-like protein, each of which has a precedented role in hydrocarbon metabolism clusters in Rhodococcus sp. and which may therefore participate in a β-myrcene degradation pathway in this organism.     

Absorption and fluorescence spectroscopic studies of the Ca2(+)-dependent lipid binding protein p36: the annexin repeat as the Ca2+ binding site

The existence of a single tryptophan residue in the protein p36, a member of a recently characterized family of Ca2+ binding proteins called annexins, is exploited to provide unique spectroscopic information on the annexin repeat motif and its role in Ca2+ binding. The differences in ultraviolet absorption and fluorescence excitation upon Ca2+ binding are interpreted solely in terms of this tryptophan, which, in view of the pronounced blue-shifts and the presence of vibronic structure, seems to reside in a highly nonpolar environment. The fluorescence emission from the protein is correspondingly blue-shifted, and it is found to transfer energy in resonance with Tb3+ absorption lines in the near-ultraviolet. This effect allows us to locate the Tb3+ and, by implication, the Ca2+ binding site to within ca. 8 A of the tryptophan residue.     

Phosphatidylinositol 4,5-bisphosphate induces actin-based movement of raft-enriched vesicles through WASP-Arp2/3

BACKGROUND: Phosphatidylinositol 4,5-bisphosphate (PIP(2)) has been implicated in the regulation of the actin cytoskeleton and vesicle trafficking. It stimulates de novo actin polymerization by activating the pathway involving the Wiskott-Aldrich syndrome protein (WASP) and the actin-related protein complex Arp2/3. Other studies show that actin polymerizes from cholesterol-sphingolipid-rich membrane microdomains called 'rafts', in a manner dependent on tyrosine phosphorylation. Although actin has been implicated in vesicle trafficking, and rafts are sites of active phosphoinositide and tyrosine kinase signaling that mediate apically directed vesicle trafficking, it is not known whether phosphoinositide regulation of actin dynamics occurs in rafts, or if it is linked to vesicle movements. RESULTS: Overexpression of type I phosphatidylinositol phosphate 5-kinase (PIP5KI), which synthesizes PIP(2), promoted actin polymerization from membrane-bound vesicles to form motile actin comets. Pervanadate (PV), a tyrosine phosphatase inhibitor, induced comets even in the absence of PIP5KI overexpression. PV increased PIP(2) levels, suggesting that it induces comets by changing PIP(2) homeostasis and by increasing tyrosine phosphorylation. Platelet-derived growth factor (PDGF) enhanced PV-induced comet formation, and these stimuli together potentiated the PIP5KI effect. The vesicles at the heads of comets were enriched in PIP5KIs and tyrosine phosphoproteins. WASP-Arp2/3 involvement was established using dominant-negative WASP constructs. Endocytic and exocytic markers identified vesicles enriched in lipid rafts as preferential sites of comet generation. Extraction of cholesterol with methyl-beta-cyclodextrin reduced comets, establishing that rafts promote comet formation. CONCLUSIONS: Sphingolipid-cholesterol rafts are preferred platforms for membrane-linked actin polymerization. This is mediated by in situ PIP(2) synthesis and tyrosine kinase signaling through the WASP-Arp2/3 pathway. Actin comets may provide a novel mechanism for raft-dependent vesicle transport and apical membrane trafficking.     

A generic algorithm for layout of biological networks

Background  

Biological networks are widely used to represent processes in biological systems and to capture interactions and dependencies between biological entities. Their size and complexity is steadily increasing due to the ongoing growth of knowledge in the life sciences. To aid understanding of biological networks several algorithms for laying out and graphically representing networks and network analysis results have been developed. However, current algorithms are specialized to particular layout styles and therefore different algorithms are required for each kind of network and/or style of layout. This increases implementation effort and means that new algorithms must be developed for new layout styles. Furthermore, additional effort is necessary to compose different layout conventions in the same diagram. Also the user cannot usually customize the placement of nodes to tailor the layout to their particular need or task and there is little support for interactive network exploration.