A novel class of microbial phosphocholine-specific phospholipases C

In this report we describe the 1,500-fold purification and characterization of the haemolytic phospholipase C (PLC) of Pseudomonas aeruginosa, the paradigm member of a novel PLC/phosphatase superfamily. Members include proteins from Mycobacterium tuberculosis, Bordetella spp., Francisella tularensis and Burkholderia pseudomallei. Purification involved overexpression of the plcHR1,2 operon, ion exchange chromatography and native preparative polyacrylamide gel electrophoresis. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry confirmed the presence of two proteins in the purified sample with sizes of 17,117.2 Da (PlcR2) and 78,417 Da (PlcH). Additionally, liquid chromatography electrospray mass spectrometry (LCMS) revealed that PlcH and PlcR2 are at a stoichiometry of 1 : 1. Western blot analysis demonstrated that the enzyme purifies as a heterodimeric complex, PlcHR2. PlcHR2 is only active on choline-containing phospholipids. It is equally active on phosphatidylcholine (PC) and sphingomyelin (SM) and is able to hydrolyse plasmenylcholine phospholipids (plasmalogens). Neither PlcHR2 nor the M. tuberculosis homologues are inhibited by D609 a widely used, competitive inhibitor of the Bacillus cereus PLC. PlcH, PlcR2, and the PlcHR2 complex bind calcium. While calcium has no detectable effect on enzymatic activity, it inhibits the haemolytic activity of PlcHR2. In addition to being required for the secretion of PlcH, the chaperone PlcR2 affects both the enzymatic and haemolytic properties of PlcH. Inclusive in these data is the conclusion that the members of this PC-PLC and phosphatase family possess a novel mechanism for the recognition and hydrolysis of their respective substrates.     

Linking central metabolism with increased pathway flux: L-valine accumulation by Corynebacterium glutamicum

Mutants of Corynebacterium glutamicum were made and enzymatically characterized to clone ilvD and ilvE, which encode dihydroxy acid dehydratase and transaminase B, respectively. These genes of the branched-chain amino acid synthesis were overexpressed together with ilvBN (which encodes acetohydroxy acid synthase) and ilvC (which encodes isomeroreductase) in the wild type, which does not excrete L-valine, to result in an accumulation of this amino acid to a concentration of 42 mM. Since L-valine originates from two pyruvate molecules, this illustrates the comparatively easy accessibility of the central metabolite pyruvate. The same genes, ilvBNCD, overexpressed in an ilvA deletion mutant which is unable to synthesize L-isoleucine increased the concentration of this amino acid to 58 mM. A further dramatic increase was obtained when panBC was deleted, making the resulting mutant auxotrophic for D-pantothenate. When the resulting strain, C. glutamicum 13032DeltailvADeltapanBC with ilvBNCD overexpressed, was grown under limiting conditions it accumulated 91 mM L-valine. This is attributed to a reduced coenzyme A availability and therefore reduced flux of pyruvate via pyruvate dehydrogenase enabling its increased drain-off via the L-valine biosynthesis pathway.     

Purification and kinetic studies on a porphobilinogen deaminase from the unicellular green alga Scenedesmus obliquus

Porphobilinogen deaminase, the enzyme condensing four molecules of porphobilinogen, was isolated and purified from light grown Scenedesmus obliquus (wild type). The purification procedure included heat treatment, ammonium sulphate fractionation, gel filtration, high-resolution anion-exchange chromatography and hydrophobic interaction chromatography. The enzyme was purified 1368-fold, compared to the initial crude extract. Its final specific activity was 6812 units · (mg · protein)^?1 at pH 7.4 with a recovery of 44%. The relative molecular mass was 33000, as determined by Sephadex G-100 gel filtration, and 35900 by lithium dodecyl sulfate-polyacrylamide-gel electrophoresis, indicating that the enzyme is a monomer. Studies of initial reaction velocities showed a linear progress curve for hydroxymethylbilane formation and a hyperbolic dependence of the initial reaction rate on substrate concentration, consistent with a sequential displacement mechanism. Apparent kinetic constants (K _m and V _max) for the conversion of porphobilinogen to hydroxymethylbilane at 37 ° C, pH 7.4, were 79 μM and 176 pmol · min^?1, respectively. Variation of both V _max and K _max with pH indicated the presence of ionizable groups in the enzyme-substrate complex(es), showing a single ionization (pK 7.15) in V _max/K _m plots. A sharp pH-profile for V _max was interpreted as a positive cooperative proton dissociation. In spite of the two pathways existing for 5-aminolevulinate biosynthesis in Scenedesmus, currently there is no indication of the existence of two porphobilinogen deaminases or even of isoenzymes.     

The occurrence, role and evolution of chromatin diminution in nematodes

Chromatin diminution takes place in presomatic cells of some parasitic nematodes during early development. This phenomenon may play an important role in somatic cell differentiation, since the somatic cells of these species undergo an extensive genome reorganization during development via chromatin diminution and polyploidization, as explained here by Clara Goday and Sergio Pimpinelli.     

The alm1+ gene from Schizosaccharomyces pombe encodes a coiled-coil protein that associates with the medial region during mitosis

We have isolated a cDNA that encodes a 142-kDa protein by immunoscreening of a Schizosaccharomyces pombe expression library with a new antibody, mAb8, that reveals spindle poles and equatorial ring-like structures in several organisms. This cDNA encodes a putative protein which we termed Alm (for abnormal long morphology). The protein is predicted to be a coiled-coil protein, containing a central α-helical domain flanked by non-helical terminal domains. Immunofluorescence analysis showed that Alm1 is localized in the medial region of the cell from anaphase to the end of cytokinesis. Cells carrying an alm1::ura4 ⁺ disruption are viable and exhibit an elongated morphology. Homozygous alm1::ura4 ⁺ diploids sporulated normally but the spores did not germinate. Spores that have inherited the disruption allele from a heterozygous alm1 ⁺ / alm1::ura4 ⁺ diploid germinated but generated smaller colonies. We propose that Alm1 participates in the structural organization of the medial region in S. pombe. Received: 9 April 1999 / Accepted: 22 October 1999     

Vanadium(V) complexes with salicylaldehyde semicarbazone derivatives bearing in vitro anti-tumor activity toward kidney tumor cells (TK-10): crystal structure of [VVO2(5-bromosalicylaldehyde semicarbazone)

As a contribution to the development of novel vanadium complexes with pharmacologically interesting moieties, new dioxovanadium(V) semicarbazone complexes with the formula cis-VO(2)L, where L=5-bromosalicylaldehyde semicarbazone and 2-hydroxynaphtalen-1-carboxaldehyde semicarbazone have been synthesized and characterized by (1)H and (13)C NMR, Raman and FTIR spectroscopies. Results were compared with those previously reported for other three analogous complexes of this series. The five complexes were tested in three different human tumor cell lines for bioactivity as potential anti-tumor agents, showing selective cytotoxicity on TK-10 cell line. Results showed that structural modifications on the semicarbazone moiety could have a significant effect on the anti-tumor activity of the vanadium complexes. In addition, the electrochemical behavior of all the complexes was studied. No apparent correlation could be demonstrated between reduction potentials of the complexes and their anti-tumor activities. The molecular structure of the novel [V(V)O(2)(5-bromosalicylaldehyde semicarbazone)] complex was solved by X-ray diffraction methods. The vanadium atom shows a distorted square pyramidal coordination sphere. The (VO(2))(+) cation is coordinated to a nearly planar (L)(-) anion acting as a tridentate ligand through both oxygen and one nitrogen atoms.     

Synthesis and biological properties of new 5-nitroindazole derivatives

A series of new 3-alkoxy- or 3-hydroxy-1-[omega-(dialkylamino)alkyl]-5-nitroindazoles have been synthesized and their trichomonacidal, antichagasic and antineoplastic properties studied. Five derivatives (5, 6, 8, 9 and 17) showed remarkable trichomonacidal activity against Trichomonas vaginalis at 10 microg/mL concentration. Three compounds (8, 10, 11) exhibited interesting antichagasic activity and these same compounds moderate antineoplastic activity against TK-10 and HT-29 cell lines. Unspecific cytotoxicity against macrophages has also been evaluated and only compounds 9, 10 and 11 resulted cytotoxic at the higher dose evaluated (100 microg/mL), loosing cytotoxicity at lower doses. QSAR studies have been carried out. X-ray crystallographic study of compound 8 has been performed.