An EXCEL-based method to search for potential Ser/Thr-phosphorylation sites in proteins

A user-friendly, inexpensive EXCEL-based program to find potential phosphorylation sites in proteins is presented. The input of the program is a protein sequence in single letter code. The program searches for 93 different protein kinase recognition sites from 30 different protein kinases. The output is a list of these sites and their position in the sequence. The program can easily be updated in case new recognition sites are described. With a few adaptations, the tool can also be used to find other patterns in a protein sequence.     

New,Non‐quinone Fluorogeldanamycin Derivatives Strongly Inhibit Hsp90

Streptomyces hygroscopicus is a natural producer of geldanamycin. Mutasynthetic supplementation of an AHBA‐blocked mutant with all possible monofluoro 3‐aminobenzoic acids provided new fluorogeldanamycins. These showed strong antiproliferative activity and inhibitory effects on human heat shock protein Hsp90. Binding to Hsp90 in the low nanomolar range was determined from molecular modelling, AFM analysis and by calorimetric studies.     

The PDE1-encoded low-affinity phosphodiesterase in the yeast Saccharomyces cerevisiae has a specific function in controlling agonist-induced cAMP signaling

The yeast Saccharomyces cerevisiae contains two genes, PDE1 and PDE2, which respectively encode a low-affinity and a high-affinity cAMP phosphodiesterase. The physiological function of the low-affinity enzyme Pde1 is unclear. We show that deletion of PDE1, but not PDE2, results in a much higher cAMP accumulation upon addition of glucose or upon intracellular acidification. Overexpression of PDE1, but not PDE2, abolished the agonist-induced cAMP increases. These results indicate a specific role for Pde1 in controlling glucose and intracellular acidification-induced cAMP signaling. Elimination of a putative protein kinase A (PKA) phosphorylation site by mutagenesis of serine252 into alanine resulted in a Pde1(ala252) allele that apparently had reduced activity in vivo. Its presence in a wild-type strain partially enhanced the agonist-induced cAMP increases compared with pde1Delta. The difference between the Pde1(ala252) allele and wild-type Pde1 was strongly dependent on PKA activity. In a RAS2(val19) pde2Delta background, the Pde1(ala252) allele caused nearly the same hyperaccumulation of cAMP as pde1Delta, while its expression in a PKA-attenuated strain caused the same reduction in cAMP hyperaccumulation as wild-type Pde1. These results suggest that serine252 might be the first target site for feedback inhibition of cAMP accumulation by PKA. We show that Pde1 is rapidly phosphorylated in vivo upon addition of glucose to glycerol-grown cells, and this activation is absent in the Pde1(ala252) mutant. Pde1 belongs to a separate class of phosphodiesterases and is the first member shown to be phosphorylated. However, in vitro the Pde1(ala252) enzyme had the same catalytic activity as wild-type Pde1, both in crude extracts and after extensive purification. This indicates that the effects of the S252A mutation are not caused by simple inactivation of the enzyme. In vitro phosphorylation of Pde1 resulted in a modest and variable increase in activity, but only in crude extracts. This was absent in Pde1(ala252), and phosphate incorporation was strongly reduced. Apparently, phosphorylation of Pde1 does not change its intrinsic activity or affinity for cAMP but appears to be important in vivo for protein-protein interaction or for targeting Pde1 to a specific subcellular location. The PKA recognition site is conserved in the corresponding region of the Schizosaccharomyces pombe and Candida albicans Pde1 homologues, possibly indicating a similar control by phosphorylation.     

Round-Robin Analysis of Anchor Bolts—Invitation

RILEM TC 90-FMA Fracture Mechanics of Concrete-ApplicationsAppendix a to Minutes from Meeting in Cardiff, Wales on September 21, 1989

Round-Robin Analysis of Anchor Bolts—Invitation     

Composition and functional analysis of the Saccharomyces cerevisiae trehalose synthase complex

In the yeast Saccharomyces cerevisiae, trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP), which convert glucose 6-phosphate plus UDP-glucose to trehalose, are part of the trehalose synthase complex. In addition to the TPS1 (previously also called GGS1, CIF1, BYP1, FDP1, GLC6, and TSS1) and TPS2 (also described as HOG2 and PFK3) gene products, this complex also contains a regulatory subunit encoded by TSL1. We have constructed a set of isogenic strains carrying all possible combinations of deletions of these three genes and of TPS3, a homologue of TSL1 identified by systematic sequencing. Deletion of TPS1 totally abolished TPS activity and measurable trehalose, whereas deletion of any of the other genes in most cases reduced both. Similarly, deletion of TPS2 completely abolished TPP activity, and deletion of any of the other genes resulted in a reduction of this activity. Therefore, it appears that all subunits are required for optimal enzymatic activity. Since we observed measurable trehalose in strains lacking all but the TPS1 gene, some phosphatase activity in addition to Tps2 can hydrolyze trehalose 6-phosphate. Deletion of TPS3, in particular in a tsl1Delta background, reduced both TPS and TPP activities and trehalose content. Deletion of TPS2, TSL1, or TPS3 and, in particular, of TSL1 plus TPS3 destabilized the trehalose synthase complex. We conclude that Tps3 is a fourth subunit of the complex with functions partially redundant to those of Tsl1. Among the four genes studied, TPS1 is necessary and sufficient for growth on glucose and fructose. Even when overproduced, none of the other subunits could take over this function of Tps1 despite the homology shared by all four proteins. A portion of Tps1 appears to occur in a form not bound by the complex. Whereas TPS activity in the complex is inhibited by Pi, Pi stimulates the monomeric form of Tps1. We discuss the possible role of differentially regulated Tps1 in a complex-bound or monomeric form in light of the requirement of Tps1 for trehalose production and for growth on glucose and fructose.     

Additive Manufacturing of Gold Nanostructures Using Nonlinear Photoreduction under Controlled Ionic Diffusion

Multiphoton photoreduction of photosensitive metallic precursors via direct laser writing (DLW) is a promising technique for the synthesis of metallic structures onto solid substrates at the sub-micron scale. DLW triggered by a two photon absorption process is done using a femtosecond NIR laser (λ = 780 nm), tetrachloroauric acid (HAuCl₄) as a gold precursor, and isinglass as a natural hydrogel matrix. The presence of a polymeric, transparent matrix avoids unwanted diffusive processes acting as a network for the metallic nanoparticles. After the writing process, a bath in deionized water removes the gold precursor ions and eliminates the polymer matrix. Different aspects underlying the growth of the gold nanostructures (AuNSs) are here investigated to achieve full control on the size and density of the AuNSs. Writing parameters (laser power, exposure time, and scanning speed) are optimized to control the patterns and the AuNSs size. The influence of a second bath containing Au³⁺ to further control the size and density of the AuNSs is also investigated, observing that these AuNSs are composed of individual gold nanoparticles (AuNPs) that grow individually. A fine-tuning of these parameters leads to an important improvement of the created structures’ quality, with a fine control on size and density of AuNSs.     

Inhibition of translation by mRNA encoding NIPP-1, a nuclear inhibitor of protein phosphatase-1

Transient transfection of COS-1 cells with an expression vector for NIPP-1, a nuclear subunit of protein phosphatase-1, did not result in an overexpression of NIPP-1 protein, although the levels of mRNA encoding NIPP-1 increased dramatically. Moreover, high concentrations of NIPP-1 mRNA inhibited the translation in reticulocyte lysates of various unrelated mRNAs. This inhibition of translation was caused by the NIPP-1 messenger and not by the translation product, since mutation of the start codon abolished NIPP-1 protein production, but had no influence on the translational inhibition. Analysis of deletion mutants showed that the inhibition was mediated by a 0.5-kb fragment in the 5'-end of the NIPP-1 mRNA. This region, when inserted in the 5'-untranslated region of the beta-galactosidase messenger, inhibited the translation of beta-galactosidase mRNA in COS-1 cells. A predicted highly stable secondary structure deltaG = -239.5 kJ/mol) is present between residues 300 and 500 of NIPP-1 mRNA. The possible importance of this structure in the translational inhibition is discussed.