Characterization of the NifU and NifS Fe-S cluster formation proteins essential for viability in Helicobacter pylori

The Fe-S cluster formation proteins NifU and NifS are essential for viability in the ulcer causing human pathogen Helicobacter pylori. Obtaining viable H. pylori mutants upon mutagenesis of the genes encoding NifU and NifS was unsuccessful even by growing the potential transformants under many different conditions including low O(2) atmosphere and supplementation with both ferric and ferrous iron. When a second copy of nifU was introduced into the chromosome at a unrelated site, creating a mero-diploid strain for nifU, this second copy of the gene could be disrupted at high frequency. This indicates that the procedures used for transformation were capable of nifU mutagenesis, so that the failure to recover mutants is solely due to the requirement of nifU for H. pylori viability. H. pylori NifU and NifS were expressed in Escherichia coli and purified to near homogeneity, and the proteins were characterized. Purified NifU is a red protein that contains approximately 1.5 atoms of iron per monomer. This iron was determined to be in the form of a redox-active [2Fe-2S](2+,+) cluster by characteristic UV-visible, EPR, and MCD spectra. The primary structure of NifU also contains the three conserved cysteine residues which are involved in providing the scaffold for the assembly of a transient Fe-S cluster for insertion into apoprotein. Purified NifS has a yellow color and UV-visible spectra characteristic of a pyridoxal phosphate containing enzyme. NifS is a cysteine desulfurase, releasing sulfur or sulfide (depending on the reducing environment) from L-cysteine, in agreement with its proposed role as a sulfur donor to Fe-S clusters. The results here indicate that the NifU type of Fe-S cluster formation proteins is not specific for maturation of the nitrogenase proteins and, as H. pylori lacks other Fe-S cluster assembly proteins, that the H. pylori NifS and NifU are responsible for the assembly of many (non-nitrogenase) Fe-S clusters.     

Identification and characterization of a eukaryotically encoded rubredoxin in a cryptomonad alga

We have identified an open reading frame with homology to prokaryotic rubredoxins (rds) on a nucleomorph chromosome of the cryptomonad alga Guillardia theta. cDNA analysis let us propose that the rd preprotein has an NH(2)-terminal extension that functions as a transit peptide for import into the plastid. Compared to rds found in non-photosynthetic prokaryotes or found in bacteria that exhibit an anoxigenic photosynthesis apparatus, nucleomorph rd has a COOH-terminal extension, which shows high homology exclusively to the COOH-termini of cyanobacterial rds as well as to a hypothetical rd in the Arabidopsis genome. This extension can be divided into a putative membrane anchor and a stretch of about 20 amino acids with unknown function linking the common rd fold to this anchor. Overexpression of nucleomorph rd in Escherichia coli using a T7 RNA polymerase/promotor system resulted in a mixture of iron-containing holorubredoxin and zinc-substituted protein. Preliminary spectroscopic studies of the iron form of nucleomorph rd suggest the existence of a native rd-type iron site. One-dimensional nuclear magnetic resonance spectroscopy of recombinant Zn-rd suggests the presence of a stable tertiary fold similar to that of other rd structures determined previously.     

Red cell enzyme polymorphisms in Friuli Venezia Giulia (northeast Italy)

Seven erythrocyte enzyme polymorphisms (ACP1, ADA, ESD, GLO1, PGD, PGM1 and PGM2) were investigated in a sample of 673 unrelated adult individuals from Friuli Venezia Giulia (or Friuli) and Istria. The gene frequencies found in the four provincial samples of Friuli and Istria fall within the range previously reported for Italy, showing a genetic homogeneity among the considered samples. However, comparisons with data from ex-Yugoslavian samples--using the chi 2 test--showed rather marked differences, probably due to a real different genetic structure of the compared samples. A significant association was found assuming a linear relation between the ADA*2 allele frequencies and longitude (r = +0.5503) and between the PGD*C frequencies and latitude (r = -0.6483), suggesting the existence of a clinal trend for these allele frequencies in Italy. These results seem to disagree with foregoing conclusions stated by other authors, probably because these studies were carried out in an area either rather narrow from the geographical point of view or affected by small size migration movements.     

Characterization of reutericyclin produced by Lactobacillus reuteri LTH2584

Lactobacillus reuteri LTH2584 exhibits antimicrobial activity that can be attributed neither to bacteriocins nor to the production of reuterin or organic acids. We have purified the active compound, named reutericyclin, to homogeneity and characterized its antimicrobial activity. Reutericyclin exhibited a broad inhibitory spectrum including Lactobacillus spp., Bacillus subtilis, B. cereus, Enterococcus faecalis, Staphylococcus aureus, and Listeria innocua. It did not affect the growth of gram-negative bacteria; however, the growth of lipopolysaccharide mutant strains of Escherichia coli was inhibited. Reutericyclin exhibited a bactericidal mode of action against Lactobacillus sanfranciscensis, Staphylococcus aureus, and B. subtilis and triggered the lysis of cells of L. sanfranciscensis in a dose-dependent manner. Germination of spores of B. subtilis was inhibited, but the spores remained unaffected under conditions that do not permit germination. The fatty acid supply of the growth media had a strong effect on reutericyclin production and its distribution between producer cells and the culture supernatant. Reutericyclin was purified from cell extracts and culture supernatant of L. reuteri LTH2584 cultures grown in mMRS by solvent extraction, gel filtration, RP-C(8) chromatography, and anion-exchange chromatography, followed by rechromatography by reversed-phase high-pressure liquid chromatography. Reutericyclin was characterized as a negatively charged, highly hydrophobic molecule with a molecular mass of 349 Da. Structural characterization (A. H?ltzel, M. G. G?nzle, G. J. Nicholson, W. P. Hammes, and G. Jung, Angew. Chem. Int. Ed. 39:2766-2768, 2000) revealed that reutericyclin is a novel tetramic acid derivative. The inhibitory activity of culture supernatant of L. reuteri LTH2584 corresponded to that of purified as well as synthetic reutericyclin.     

Increased expression of mature cathepsin B in aging rat liver

Senescence has been proposed as an important safeguard against neoplasia. One of the hallmarks of cellular senescence in vitro as well as human aging in vivo is a reduced intracellular protein catabolism. The pathways affected and the mechanisms responsible for the decrease in overall protein turnover in aging cells are not well understood. Our aim was to determine whether or not expression of one of the major hepatic lysosomal cysteine peptidases, cathepsin B, changes during aging of Sprague-Dawley rats. Cathepsin B activity was assessed in whole rat liver homogenates, and was found to be increased fourfold (P< or =0.001) in aged livers compared with younger counterparts. This was paralleled by an at least a twofold increase in mature cathepsin B protein. Nonetheless, Northern blot analysis of total liver RNA revealed no change in steady-state levels of cathepsin B mRNAs. These findings seem to contradict the present dogma according to which aging tissues have a reduced intracellular capacity to catabolise proteins. We propose that our earlier observation of the accumulation of T-kininogen, a potent but reversible cysteine peptidase inhibitor, in aging rat liver may provide a plausible explanation for this discrepancy.     

Evidence for sequential action of cdc7 and cdk2 protein kinases during initiation of DNA replication in Xenopus egg extracts

To investigate how the protein kinase cdc7 stimulates DNA replication in metazoans, a soluble cell-free replication system derived from Xenopus eggs was used. DNA was incubated in egg cytosol to form prereplication complexes and then in nucleoplasmic extract to initiate DNA synthesis. We find that cdc7 is greatly enriched in nucleoplasmic extract and that this high concentration is essential for efficient DNA replication, supporting previous models that the nucleus activates replication indirectly by sequestering essential components. cdc7 binds to chromatin at the G(1)/S transition before initiation occurs, and it dissociates from chromatin as S phase progresses. The chromatin association of cdc7 requires chromatin-bound MCM. In turn, cdc7 is required to load the initiation factor cdc45 onto the DNA. Finally, efficient replication is observed when chromatin is exposed first to cdc7 and then to cdk2 but not when it is exposed to cdk2 before cdc7. Therefore, the cdc7- and cdk2-dependent initiation steps can be separated, indicating the existence of a novel, stable initiation intermediate. Moreover, the data suggest that cdk2 can only act after cdc7 has executed its function.     

Constitutive phosphorylation of the Parkinson's disease associated alpha-synuclein

alpha-Synuclein has been implicated in the pathogenesis of Parkinson's disease, since rare autosomal dominant mutations are associated with early onset of the disease and alpha-synuclein was found to be a major constituent of Lewy bodies. We have analyzed alpha-synuclein expression in transfected cell lines. In pulse-chase experiments alpha-synuclein appeared to be stable over long periods (t((1)/(2)) 54 h) and no endoproteolytic processing was observed. alpha-Synuclein was constitutively phosphorylated in human kidney 293 cells as well as in rat pheochromocytoma PC12 cells. In both cell lines phosphorylation was highly sensitive to phosphatases, since okadaic acid markedly stabilized phosphate incorporation. Phosphoamino acid analysis revealed that phosphorylation occurred predominantly on serine. Using site-directed mutagenesis we have identified a major phosphorylation site at serine 129 within the C-terminal domain of alpha-synuclein. An additional site, which was phosphorylated less efficiently, was mapped to serine 87. The major phosphorylation site was located within a consensus recognition sequence of casein kinase 1 (CK-1). In vitro experiments and two-dimensional phosphopeptide mapping provided further evidence that serine 129 was phosphorylated by CK-1 and CK-2. Moreover, phosphorylation of serine 129 was reduced in vivo upon inhibition of CK-1 or CK-2. These data demonstrate that alpha-synuclein is constitutively phosphorylated within its C terminus and may indicate that the function of alpha-synuclein is regulated by phosphorylation/dephosphorylation.