Purification and characterization of recombinant microsomal prostaglandin E synthase-1

Recombinant human microsomal prostaglandin E(2) synthase-1 (mPGES-1) was expressed in a baculovirus-Sf9 cell system. The mPGES-1 was solubilized from Sf9 cell membranes with diheptanoylphosphatidylcholine and purified in the presence of octylglucoside using hydroxyapatite column chromatography. The K(m) values of the substrates PGH(2) and GSH were 14 microM and 0.75 mM, respectively, with the purified enzyme. The specific activity (4 micromol/min/mg) was increased 3-5-fold by non-ionic and zwitterionic detergents. Kinetic analysis showed that dodecylmaltoside increases V(max) but does not affect the K(m) values of either substrate. Several other thiol-containing compounds were tested as glutathione replacements, none of which yielded detectable enzyme activity. During enzyme catalysis, glutathione was not oxidized and therefore can be considered an enzyme cofactor. No glutathione transferase or peroxidase activity could be determined with a range of potential substrates. The results show that purified mPGES-1 has a specific activity similar to Cox-2, consistent with its postulated role in Cox-2 mediated PGE(2) formation.     

Enhancement of poplar glutaredoxin expression by optimization of the cDNA sequence

Glutaredoxins are low-molecular-weight oxidore ductases that play an important role in redox regulation in eukaryotic and prokaryotic cells. Because of their low abundance, these proteins are poorly characterized in plants. Furthermore, very poor yields have been obtained with the expression systems prepared so far, and in addition, the recombinant products contain a His-tag which can interfere with the biochemical characterization. In order to obtain more information about those important regulatory proteins in plants, a cDNA coding for an extended glutaredoxin has been introduced into the expression plasmid pET-3d and the resulting construction has been used to transform Escherichia coli strain BL21(DE3) in the presence of plasmid helper pSBET or not. Initially poor or ineffective protein expression has been improved by successively cloning a N-terminus truncated form of the protein, introducing silent mutations both at the 5' and at the 3' ends of the nucleotide sequence, and finally altering the 3' end in order to change the C-terminus amino acid sequence of the protein. The first modifications have allowed us to produce the protein in large amounts but essentially in an insoluble form which could be resolubilized and purified. On the other hand, changing the C-terminus sequence resulted in protein preparations of high purity and in a soluble form. The recombinant proteins were biochemically active and the yield varied between 6 and 14 mg of homogeneous protein per liter of culture.     

Patterns of importin-alpha expression during Drosophila spermatogenesis

Importin-alpha proteins do not only mediate the nuclear import of karyophilic proteins but also regulate spindle assembly during mitosis and the assembly of ring canals during Drosophila oogenesis. Three importin-alpha genes are present in the genome of Drosophila. To gain further insights into their function we analysed their expression during spermatogenesis by using antibodies raised against each of the three Importin-alpha proteins identified in Drosophila, namely, Imp-alpha1, -alpha2, and -alpha3. We found that each Imp-alpha is expressed during a specific and limited period of spermatogenesis. Strong expression of Imp-alpha2 takes place in spermatogonial cells, persists in spermatocytes, and lasts up to the completion of meiosis. In growing spermatocytes, the intracellular localisation of Imp-alpha2 appears to be dependent upon the rate of cell growth. In pupal testes Imp-alpha2 is essentially present in the spermatocyte nucleus but is localised in the cytoplasm of spermatocytes from adult testes. Both Imp-alpha1 and -alpha3 expression initiates at the beginning of meiosis and ends during spermatid differentiation. Imp-alpha1 expression extends up to the onset of the elongation phase, whereas that of Imp-alpha3 persists up to the completion of nuclear condensation when the spermatids become individualised. During meiosis Imp-alpha1 and -alpha3 are dispersed in the karyoplasm where they are partially associated with the nuclear spindle, albeit not with the asters. At telophase they aggregate around the chromatin. During sperm head differentiation, both Imp-alpha1 and -alpha3 are nuclear. These data indicate that each Imp-alpha protein carries during Drosophila spermatogenesis distinct, albeit overlapping, functions that may involve nuclear import of proteins, microtubule organisation, and other yet unknown processes.     

Structure-based design of a potent purine-based cyclin-dependent kinase inhibitor

Aberrant control of cyclin-dependent kinases (CDKs) is a central feature of the molecular pathology of cancer. Iterative structure-based design was used to optimize the ATP- competitive inhibition of CDK1 and CDK2 by O(6)-cyclohexylmethylguanines, resulting in O(6)-cyclohexylmethyl-2-(4'- sulfamoylanilino)purine. The new inhibitor is 1,000-fold more potent than the parent compound (K(i) values for CDK1 = 9 nM and CDK2 = 6 nM versus 5,000 nM and 12,000 nM, respectively, for O(6)-cyclohexylmethylguanine). The increased potency arises primarily from the formation of two additional hydrogen bonds between the inhibitor and Asp 86 of CDK2, which facilitate optimum hydrophobic packing of the anilino group with the specificity surface of CDK2. Cellular studies with O(6)-cyclohexylmethyl-2-(4'- sulfamoylanilino) purine demonstrated inhibition of MCF-7 cell growth and target protein phosphorylation, consistent with CDK1 and CDK2 inhibition. The work represents the first successful iterative synthesis of a potent CDK inhibitor based on the structure of fully activated CDK2-cyclin A. Furthermore, the potency of O(6)-cyclohexylmethyl-2-(4'- sulfamoylanilino)purine was both predicted and fully rationalized on the basis of protein-ligand interactions.     

Phylogenetic classification of transporters and other membrane proteins from Saccharomyces cerevisiae

On the basis of functional and phylogenetic criteria, we have identified a total of 229 subfamilies and 111 singletons predicted to carry out transport or other membrane functions in Saccharomyces cerevisiae. We have extended the Transporter Classification (TC) and created a Membrane Classification (MC) for non-transporter membrane proteins. Using the preliminary phylogenetic digits X, Y, Z (for new families, subfamilies, and clusters, respectively), we allocated a five-digit number to 850 proteins predicted to contain more than two transmembrane domains. Compared with a previous TC of the yeast genome, we classified an additional set of 538 membrane proteins (transporters and non-transporters) and identified 111 novel phylogenetic subfamilies. Electronic Publication     

Infrared imaging of 2-D temperature distribution during cryogen spray cooling

Cryogen spray cooling (CSC) is used in conjunction with pulsed laser irradiation for treatment of dermatologic indications. The main goal of this study was to determine the radial temperature distribution created by CSC and evaluate the importance of radial temperature gradients upon the subsequent analysis of tissue cooling throughout the skin. Since direct measurement of surface temperatures during CSC are hindered by the formation of a liquid cryogen layer, temperature distributions were estimated using a thin, black aluminum sheet. An infrared focal plane array camera was used to determine the 2-D backside temperature distribution during a cryogen spurt, which preliminary measurements have shown is a good indicator of the front-side temperature distribution. The measured temperature distribution was approximately gaussian in shape. Next, the transient temperature distributions in skin were calculated for two cases: 1) the standard 1-D solution which assumes a uniform cooling temperature distribution, and 2) a 2-D solution using a nonuniform surface cooling temperature distribution based upon the back-side infrared temperature measurements. At the end of a 100-ms cryogen spurt, calculations showed that, for the two cases, large discrepancies in temperatures at the surface and at a 60-micron depth were found at radii greater than 2.5 mm. These results suggest that it is necessary to consider radial temperature gradients during cryogen spray cooling of tissue.     

Insight into the chiral recognition of warfarin enantiomers by epichlorhydrin/beta-cyclodextrin polymer-based supports: determination of stoichiometry and stability of warfarin/beta-cyclodextrin polymer complexes

The complexation of warfarin (W) enantiomers by a hydrosoluble high-molecular-weight beta-cyclodextrin/epichlohydrin polymer (EP/beta-CD polymer) was studied using HPLC with a mobile phase of methanol/0.1 M Na acetate/acetic acid (pH 4) at 22 degrees C. It was found that the complexes (W/beta-CD unit) have a 1:1 stoichiometry. The stability constants of the complex involving each enantiomer and the polymer beta-CD units were determined in the mobile phase, and the highest stability of the complex (S-warfarin/beta-CD unit) was observed. From the chromatographic separations of warfarin enantiomers on different beta-CD or its derivative supports, we have deduced the role of the simultaneous presence of several glyceryl (-O-CH(2)-CHOH-CH(2)-O-) and dihydroxypropyl (-O-CH(2)-CHOH-CH(2)OH) groups on one beta-CD ring in promoting the chiral recognition of warfarin enantiomers.     

Glycogen metabolism loss: a common marker of parasitic behaviour in bacteria?

We searched 55 completely sequenced bacterial genomes for glycogen synthesis and degradation enzymes. A significant proportion of these bacteria appears to lack glycogen metabolism capability. Interestingly, these bacteria are parasitic, symbiotic or fastidious (i.e. difficult to culture outside their normal environment). It is suggested that the lack of bacterial glycogen metabolism is a trait associated with parasitic behaviour in bacteria.