A tomato enzyme catalyzing the phosphorylation of 3,4-dihydroxy-2-butanone

A riboflavin biosynthesis ribB mutant of Escherichia coli deficient of 3,4-dihydroxy-2-butanone 4-phosphate synthase was complemented with a cDNA library from Lycopersicon esculentum. The complementing gene was isolated and expressed in E. coli. The resulting protein was shown to specify a 62 kDa protein which phosphorylates dihydroxyacetone, both enantiomers of 3,4-dihydroxy-2-butanone, and several other aldoses and ketoses. Sequence analysis revealed homology to dihydroacetone kinases (dak) genes from plants, animals, fungi and some eubacteria. Genes with similarity to the 5' part of the dak gene from tomato were found in many other eubacteria. The physiological role of the dak gene is still incompletely known.     

Quantitative high-performance liquid chromatography/electrospray ionization tandem mass spectrometric analysis of 2- and 3-series prostaglandins in cultured tumor cells

This paper describes a rapid and simple technique for the simultaneous quantitative analysis of PGE(2), PGE(3), and other closely related prostaglandins from cultured cells using liquid chromatography/electrospray ionization tandem mass spectrometry. This method permits quantification of selected individual prostaglandins derived either from arachidonic acid (AA) or eicosapentaenoic acid (EPA) from cell extracts without tedious derivatization, lengthy sample preparation, and separation required by GC-MS- or HPLC-UV-based methods. The validation assessment showed that the quantitative determination is linear (r(2)>0.999) for both PGE(2) and PGE(3) in the range tested (1-500 ng/ml, 0.0028-1.4 microM) and a coefficient of variation lower than 10% was obtained for samples analyzed on 3 separate days. The detection limit was 2.5 pg for both PGE(2) and PGE(3). Extraction efficiency of PGE(2) and PGE(3) from cell suspensions ranged from 89.4 to 98.2%. As an application of the method, prostaglandins formed by EPA in human lung cancer A549 cells were determined. A 62% reduction of PGE(2) formation was noted when A549 cells were treated with 10 microM of EPA. Concomitantly, EPA increased formation of PGE(3) by 10-fold in A549 cells. This is the first report that unequivocally demonstrates that EPA can be converted to PGE(3) by cyclooxygenase in human cancer cells.     

Alpha-helix structure in Alzheimer's disease aggregates of tau-protein

The discovery of beta-sheet structure in Alzheimer's amyloid fibrils, and then in many other disease-related protein fibrils, has led to the widely believed view that beta-sheet formation is the general mechanism of aberrant protein aggregation leading to disease. This notion is further reinforced by recent findings, which indicate that normal proteins can be induced to form beta-sheet fibrils in vitro. Alzheimer's disease, a paradigm proteopathy, is accompanied by the formation of two distinct aggregates, amyloid fibrils and paired helical filaments (PHFs). Electron microscope images of PHFs show pairs of twisted ribbons with 80 nm periodicity. However, there is little information of the molecular structure of PHFs, as previous studies have failed to identify signs of regular structure. Using far-UV circular dichroism and Fourier-transformed infrared spectroscopy, we find that PHFs are comprised of alpha-helices. This is remarkable as tau-protein, PHF's primary constituent, has a high abundance of helix-breaking amino acids and is unstructured in solution. We also find that PHFs are very stable, as judged by their high melting temperature and resistance to protease digestion. PHFs are the first example of pathological aggregation associated to the formation of alpha-helix.     

Characterization of Surfactin-like Cyclic Depsipeptides Synthesized by Bacillus pumilus from Ascidian Halocynthia aurantium

A marine bacterium (KMM 1364), identified as Bacillus pumilus, was isolated from the surface of ascidian Halocynthia aurantium. Structural analysis revealed that the strain KMM 1364 produced a mixture of lipopeptide surfactin analogs with major components with molecular masses of 1035, 1049, 1063, and 1077. The variation in molecular weight represents changes in the number of methylene groups in the lipid and/or peptide portions of the compounds. Structurally, these lipopeptides differ from surfactin in the substitution of the valine residue in position 4 by leucine, and have been isolated as two carboxy-terminal variants, with valine or isoleucine in position 7. As constituents of the lipophilic part of the peptides, only β-hydroxy-C₁₅-, β-hydroxy-C₁₆-, and a high amount of β-hydroxy-C₁₇ fatty acid were determined.     

UL27.5 Is a Novel γ2 Gene Antisense to the Herpes Simplex Virus 1 Gene Encoding Glycoprotein B

An antibody made against the herpes simplex virus 1 U_S5 gene predicted to encode glycoprotein J was found to react strongly with two proteins, one with an apparent M_r of 23,000 and mapping in the S component and one with a herpes simplex virus protein with an apparent M_r of 43,000. The antibody also reacted with herpes simplex virus type 2 proteins forming several bands with apparent M_rs ranging from 43,000 to 50,000. Mapping studies based on intertypic recombinants, analyses of deletion mutants, and ultimately, reaction of the antibody with a chimeric protein expressed by in-frame fusion of the glutathione S-transferase gene to an open reading frame antisense to the gene encoding glycoprotein B led to the definitive identification of the new open reading frame, designated U_L27.5. Sequence analyses indicate the conservation of a short amino acid sequence common to U_S5 and U_L27.5. The coding sequence of the herpes simplex virus U_L27.5 open reading frame is strongly homologous to the sequence encoding the carboxyl terminus of the herpes simplex virus 2 U_L27.5 sequence. However, both open reading frames could encode proteins predicted to be significantly larger than the mature U_L27.5 proteins accumulating in the infected cells, indicating that these are either processed posttranslationally or synthesized from alternate, nonmethionine-initiating codons. The U_L27.5 gene expression is blocked by phosphonoacetate, indicating that it is a γ₂ gene. The product accumulated predominantly in the cytoplasm. U_L27.5 is the third open reading frame found to map totally antisense to another gene and suggests that additional genes mapping antisense to known genes may exist.     

Breakdown of Ficus and Eucalyptus leaves in an organically polluted river in India: fungal diversity and ecological functions

1. At two organically polluted sites in the River Nethravathi, banyan and eucalypt leaves were colonized by one or two species of aquatic hyphomycetes. A total of three or four species were identified at the two sites in samples of water and naturally occurring leaves.
2. Spore production from stream‐exposed leaves by aquatic hyphomycetes was lower by a factor of up to 1 million compared with an earlier study in geographically close but unpolluted streams.
3. Exponential decay rates and loss rates of phosphorus and calcium, were not statistically different from an earlier study in unpolluted streams. Nitrogen increased during decomposition more slowly in the current study.
4. The microbial community on both leaves released enzymes active against starch, pectin, cellulose and xylan.
5. Banyan leaves conditioned for 12 weeks were more palatable to the gastropod Notopala sp. than unconditioned leaves.
6. Together with earlier data from unpolluted streams, the study provides evidence that organic pollution severely restricts diversity of aquatic hyphomycetes and their reproductive output, but does not have an equally strong effect on ecological functions generally associated with this group.     

Functional genomics by NMR spectroscopy. Phenylacetate catabolism in Escherichia coli.

Aerobic metabolism of phenylalanine in most bacteria proceeds via oxidation to phenylacetate. Surprisingly, the further metabolism of phenylacetate has not been elucidated, even in well studied bacteria such as Escherichia coli. The only committed step is the conversion of phenylacetate into phenylacetyl-CoA. The paa operon of E. coli encodes 14 polypeptides involved in the catabolism of phenylacetate. We have found that E. coli K12 mutants with a deletion of the paaF, paaG, paaH, paaJ or paaZ gene are unable to grow with phenylacetate as carbon source. Incubation of a paaG mutant with [U-13C8]phenylacetate yielded ring-1,2-dihydroxy-1,2-dihydrophenylacetyl lactone as shown by NMR spectroscopy. Incubation of the paaF and paaH mutants with phenylacetate yielded delta3-dehydroadipate and 3-hydroxyadipate, respectively. The origin of the carbon atoms of these C6 compounds from the aromatic ring was shown using [ring-13C6]phenylacetate. The paaG and paaZ mutants also converted phenylacetate into ortho-hydroxyphenylacetate, which was previously identified as a dead end product of phenylacetate catabolism. These data, in conjunction with protein sequence data, suggest a novel catabolic pathway via CoA thioesters. According to this, phenylacetyl-CoA is attacked by a ring-oxygenase/reductase (PaaABCDE proteins), generating a hydroxylated and reduced derivative of phenylacetyl-CoA, which is not re-oxidized to a dihydroxylated aromatic intermediate, as in other known aromatic pathways. Rather, it is proposed that this nonaromatic intermediate CoA ester is further metabolized in a complex reaction sequence comprising enoyl-CoA isomerization/hydration, nonoxygenolytic ring opening, and dehydrogenation catalyzed by the PaaG and PaaZ proteins. The subsequent beta-oxidation-type degradation of the resulting CoA dicarboxylate via beta-ketoadipyl-CoA to succinyl-CoA and acetyl-CoA appears to be catalyzed by the PaaJ, PaaF and PaaH proteins.     

Method of non-destructive mechanical testing of new surface coatings for prostheses]

Research into new surface coatings and surface processing methods for prostheses is subject to numerous studies. The aim of this study was to test an innovative biomechanical measuring method for the examination of the ingrowth of bone implants. Using a transcortical model, coated (n=14) or uncoated (n=14) titanic cylinders were implanted into the lateral condyle of 28 New Zealand White Rabbits. After 6 weeks or 6 months the animals were sacrificed and the osseointegration of the implants was evaluated biomechanically and histologically. Up to traction of 50 N the load dependent movement between bone and testing cylinder did not lead to a destruction of the bone-implant-interface. Therefore, biomechanical and histological investigations could be performed in the same specimen. The results of both evaluations showed a significant correlation (correlation coefficient -0.79; p < 0.01) and were absolutely reproducible. With the method of non-destructive mechanical testing, it is possible to halve the number of required animals. Additionally, the results of the biomechanical and histological analysis can be compared and thus serve as an internal control. In summary, the method of non-destructive mechanical testing represents an ideal tool to study new surface coatings and surface processing methods for prostheses.