Amaranth Globulin Polypeptide Heterogeneity

The polypeptides integrating amaranth globulin-p and 11S-globulin were characterized by two-dimensional electrophoresis, ion-exchange chromatography and RP-HPLC. All polypeptides exhibited charge and hydrophobic heterogeneity. Almost all acid (A, pI 5–7) and basic (B, pI 9–10) polypeptides were present in both globulins, and the same happened with the unprocessed M polypeptides with pI in the range of 7–7.5 which fits well with a sequence containing both the A and B polypeptides. There were other polypeptides only present in 11S-globulin, like some of 41 and 16 kDa, which might come from another precursor or be the products of a different processing of the propolypeptide. These results suggested that, although amaranth subunits from different subfamilies are interchangeable in different oligomers, some structural differences between them might affect the assembly of globulin molecules. Structural differences arising from this behavior could account for the different physicochemical properties of globulin molecules.     

Biochemical characterization and structural prediction of a novel cytosolic leucyl aminopeptidase of the M17 family from Schizosaccharomyces pombe

A new leucyl aminopeptidase activity has been identified in the fission yeast Schizosaccharomyces pombe. The enzyme, which has been purified and named leucyl aminopeptidase yspII (LAP yspII), had a molecular mass of 320 and 54 kDa by gel filtration and SDS/PAGE, respectively, suggesting a homohexameric structure. The enzyme cleaved synthetic aminoacyl-4-nitroanilides at an optimum of pH 8.5, and preferred leucine and methionine as N-terminal amino acids. A clear dependence on Mn2+ concentration for activity was found, and an apparent association constant of 0.33 mM was calculated for the metal ion. Bestatin behaved as a competitive inhibitor of LAP yspII (K(i) = 0.14 microM), while chelating agents such as chloroquine, EDTA and 1,10-phenanthroline also reduced enzyme activity. A MALDI-MS analysis, followed by sequencing of two of the resulting peptides, showed that LAP yspII undoubtedly corresponds to the putative aminopeptidase C13A11.05 identified in the S. pombe genome project. The protein exhibited nearly 40% sequence identity to fungal and mammalian aminopeptidases belonging to the M17 family of metallopeptidases. Catalytic residues (Lys292 and Arg366), as well as those involved in coordination with the cocatalytic metal ions (Lys280, Asp285, Asp303, Asp362 and Glu364) and those forming the hydrophobic pocket for substrate binding (Met300, Asn360, Ala363, Thr390, Leu391, Ala483 and Met486), were perfectly conserved among all known aminopeptidases. The S. pombe enzyme is predicted to be formed two clearly distinguished domains with a well conserved C-terminal catalytic domain showing a characteristic topology of eight beta-sheets surrounded by alpha-helical segments in the form of a saddle.     

Precision and accuracy of Dahl-Lea back-calculated smolt lengths from adult scales of Atlantic salmon Salmo salar

Using tagged and recaptured Atlantic salmon Salmo salar (n = 106) the present analysis shows that the most commonly applied linear back-calculation method for estimating past length, the Dahl-Lea method, resulted in overestimation of the length of large smolts and underestimation of small smolts. A correction equation (y = 0.53x + 6.23) for estimating true smolt length (y) from lengths back-calculated from adult scale measures (x) to account for these systematic discrepancies is proposed.     

Genetics of osteopontin in patients with chronic kidney disease: The German Chronic Kidney Disease study

Osteopontin (OPN), encoded by SPP1, is a phosphorylated glycoprotein predominantly synthesized in kidney tissue. Increased OPN mRNA and protein expression correlates with proteinuria, reduced creatinine clearance, and kidney fibrosis in animal models of kidney disease. But its genetic underpinnings are incompletely understood. We therefore conducted a genome-wide association study (GWAS) of OPN in a European chronic kidney disease (CKD) population. Using data from participants of the German Chronic Kidney Disease (GCKD) study (N = 4,897), a GWAS (minor allele frequency [MAF]≥1%) and aggregated variant testing (AVT, MAF<1%) of ELISA-quantified serum OPN, adjusted for age, sex, estimated glomerular filtration rate (eGFR), and urinary albumin-to-creatinine ratio (UACR) was conducted. In the project, GCKD participants had a mean age of 60 years (SD 12), median eGFR of 46 mL/min/1.73m² (p25: 37, p75: 57) and median UACR of 50 mg/g (p25: 9, p75: 383). GWAS revealed 3 loci (p<5.0E-08), two of which replicated in the population-based Young Finns Study (YFS) cohort (p<1.67E-03): rs10011284, upstream of SPP1 encoding the OPN protein and related to OPN production, and rs4253311, mapping into KLKB1 encoding prekallikrein (PK), which is processed to kallikrein (KAL) implicated through the kinin-kallikrein system (KKS) in blood pressure control, inflammation, blood coagulation, cancer, and cardiovascular disease. The SPP1 gene was also identified by AVT (p = 2.5E-8), comprising 7 splice-site and missense variants. Among others, downstream analyses revealed colocalization of the OPN association signal at SPP1 with expression in pancreas tissue, and at KLKB1 with various plasma proteins in trans, and with phenotypes (bone disorder, deep venous thrombosis) in human tissue. In summary, this GWAS of OPN levels revealed two replicated associations. The KLKB1 locus connects the function of OPN with PK, suggestive of possible further post-translation processing of OPN. Further studies are needed to elucidate the complex role of OPN within human (patho)physiology.     

Laser pyrolysis products-genotoxic, clastogenic and mutagenic effects of the particulate aerosol fractions

Laser therapy has gained wide acceptance and application in many medical disciplines. Nevertheless, during surgical procedures, the thermal destruction of tissue creates a smoke plume. Recent research data indicate that pyrolysates liberated during vaporisation of tissue induce DNA damage. However, assessing potential health hazards during medical laser treatment requires comprehensive insight into the cytotoxic, genotoxic, clastogenic and mutagenic capacity of laser pyrolysis products (LPP). Therefore, the aim of this study was to evaluate the cytotoxic, genotoxic, clastogenic and mutagenic potential of substances resulting from laser irradiation. Four different types of porcine tissues were irradiated with a surgical CO2 laser, the aerosols were sampled under defined conditions and subjected to the SCE test, micronucleus test and the HPRT test. The results showed that the pyrolysis products are strong inducers of cytotoxic effects. The pyrolysis products induced positive effects in the SCE test, micronucleus test and the HPRT test. The ability and extent to induce genotoxic and mutagenic effects turned out to be dependent on the type of tissue that had been irradiated. In general, the effects were most pronounced with liver pyrolysate. In all test systems, a clear dose relationship could be established. In conclusion, we were able to prove that the particulate fraction of laser pyrolysis aerosols originating from biological tissues undoubtedly have to be classified as cytotoxic, genotoxic, clastogenic and mutagenic. Therefore, they could be potential health hazards for humans.     

Kinetic study of phenol hydroxylase and catechol 1,2-dioxygenase biosynthesis by <Emphasis Type="Italic">Candida tropicalis</Emphasis> cells grown on different phenolic substrates

When Candida tropicalis was grown on phenol, catechol or resorcinol, the highest levels of specific activity of phenol hydroxylase (EC. 1.14.13.7) and catechol 1,2-dioxygenase (EC. 1.13.11.1) were attained with phenol. With the three aromatic compounds tested, the yeast cells exhibited sharp peaks of specific activity of both enzymes at particular incubation times. Phenol-induced cells containing high levels of both enzymes were capable of degrading rapidly and without delay 4-chlorophenol and 2,6-dichlorophenol, and to a lesser extend pentachlorophenol. However, the yeast could not grow on chlorophenols as major carbon and energy source.     

Peroxisomal localization of sulfite oxidase separates it from chloroplast-based sulfur assimilation

Recently, we isolated the sulfite oxidase (SO) gene from Arabidopsis thaliana and characterized the purified SO protein. The purpose of the present study was to determine the subcellular localization of this novel plant enzyme. Immunogold electron-microscopic analysis showed the gold labels nearly exclusively in the peroxisomes. To verify this finding, green fluorescent protein was fused to full-length plant SO including the putative peroxisomal targeting signal 1 (PTS1) 'SNL' and expressed in tobacco leaves. Our results showed a punctate fluorescence pattern resembling that of peroxisomes. Co-labelling with MitoTracker-Red excluded that the observed fluorescence was due to mitochondrial sorting. By investigation of deleted or mutated PTS1, no functional peroxisomal targeting signal 2 (PTS2) could be detected in plant SO. This conclusion is supported by expression studies in Pichia pastoris mutants with defined defects either in PTS1- or PTS2-mediated peroxisomal import.     

Rapid detection and enumeration of Legionella pneumophila in hot water systems by solid-phase cytometry

A new method for the rapid and sensitive detection of Legionella pneumophila in hot water systems has been developed. The method is based on an IF assay combined with detection by solid-phase cytometry. This method allowed the enumeration of L. pneumophila serogroup 1 and L. pneumophila serogroups 2 to 6, 8 to 10, and 12 to 15 in tap water samples within 3 to 4 h. The sensitivity of the method was between 10 and 100 bacteria per liter and was principally limited by the filtration capacity of membranes. The specificity of the antibody was evaluated against 15 non-Legionella strains, and no cross-reactivity was observed. When the method was applied to natural waters, direct counts of L. pneumophila were compared with the number of CFU obtained by the standard culture method. Direct counts were always higher than culturable counts, and the ratio between the two methods ranged from 1.4 to 325. Solid-phase cytometry offers a fast and sensitive alternative to the culture method for L. pneumophila screening in hot water systems.