Hydrolysis of alpha-human atrial natriuretic peptide in vitro by human kidney membranes and purified endopeptidase-24.11. Evidence for a novel cleavage site.

alpha-Human atrial natriuretic peptide (hANP) is secreted by the heart and acts on the kidney to promote a strong diuresis and natriuresis. In vivo it has been shown to be catabolized partly by the kidney. Crude microvillar membranes of human kidney degrade 125I-ANP at several internal bonds generating metabolites among which the C-terminal fragments were identified. Formation of the C-terminal tripeptide was blocked by phosphoramidon, indicating the involvement of endopeptidase-24.11 in this cleavage. Subsequent cleavages by aminopeptidase(s) yielded the C-terminal dipeptide and free tyrosine. Using purified endopeptidase 24.11, we identified seven sites of hydrolysis in unlabelled alpha-hANP: the bonds Arg-4-Ser-5, Cys-7-Phe-8, Arg-11-Met-12, Arg-14-Ile-15, Gly-16-Ala-17, Gly-20-Leu-21 and Ser-25-Phe-26. However, the bonds Gly-16-Ala-17 and Arg-4-Ser-5 did not fulfil the known specificity requirements of the enzyme. Cleavage at the Gly-16-Ala-17 bond was previously observed by Stephenson & Kenny [(1987) Biochem. J. 243, 183-187], but this is the first report of an Arg-Ser bond cleavage by this enzyme. Initial attack of alpha-hANP by endopeptidase-24.11 took place at a bond within the disulphide-linked loop and produced a peptide having the same amino acid composition as intact ANP. The bond cleaved in this metabolite was determined as the Cys-7-Phe-8 bond. Determination of all the bonds cleaved in alpha-hANP by endopeptidase-24.11 should prove useful for the design of more stable analogues, which could have therapeutic uses in hypertension.     

Yeast species utilizing uric acid,adenine,n-alkylamines or diamines as sole source of carbon and energy

Yeast strains utilizing uric acid, adenine, monoamines or diamines as sole source of carbon and energy were isolated from several soil samples by the enrichment culture method. The most common species wasTrichosporon cutaneum. Strains ofCandida catenulata, C. famata, C. parapsilosis, C. rugosa, Cryptococcus laurentii, Stephanoascus ciferrii andTr. adeninovorans were also isolated. All strains utilizing uric acid as sole carbon source utilized some primaryn-alkyl-l-amines hydroxyamines or diamines as well. The ascomycetous yeast strains showing these characteristics all belonged to species known to assimilate hydrocarbons. Type strains of hydrocarbon-positive yeast species which were not found in the enrichment cultures generally assimilated putrescine, some type strains also butylamine or pentylamine, but none assimilated uric acid. Methanol-positive species were not isolated. Type strains of methanol-positive and of hydrocarbon-negative species did not assimilate uric acid, butylamine or putrescine. Assimilation of putrescine as sole source of carbon and energy may be a valuable diagnostic criterion in yeast taxonomy.     

Cytological characterization of isolated sperm cells of perennial ryegrass (Lolium perenne L.)

Summary The cytoplasmic content and the distribution of intramembrane particles (IMPs) of the plasma membrane of isolated sperm cells of perennial ryegrass (Lolium perenne L.) have been characterized using flow cytometry, transmission electron microscopy, confocal scanning laser microscopy and freeze-fracture studies. The isolated haploid sperm cells contain a variety of cell organelles with the exception of microtubules. Proplastids and plastids with starch were observed, although only rarely. Vacuoles containing remnants of organelles and stacked lamellae of endoplasmic reticulum with cytoplasmic inclusions were observed frequently, indicating that autophagy takes place. The number of mitochondria varies from 11 to 26 with an average of 17. Generally, the nucleus has a lobed shape and displays various interphasic stages of chromatin condensation. The analysis of the number of mitochondria and the nuclear state did not show evidence of sperm cell dimorphism. The cytological variability observed, could be explained by differences in developmental stages already present in vivo at the moment of isolation. No correlation between the number of mitochondria and the nuclear cross-sectioned area and/or the condensation state of the chromatin could be found. The density of intramembrane particles of the plasma membrane on the exoplasmic fracture face is more than twice that on the protoplasmic fracture face. That is the opposite of what was found for sporophytic cells of perennial ryegrass. These results are discussed in relation to the potential use of these cells for biotechnology and developmental studies.     

Iron content of sediment and phosphate adsorption properties

Phosphorus can occur in sediments in different forms and accordingly its availability varies. The distinction between the phosphorus fractions is made with two chemical extraction methods; an ammonium oxalate-oxalic acid extraction and an extraction according to Hieltjes & Lijklema (1980).The iron and aluminum liberated with the ammonium oxalate-oxalic acid extraction method is linearly correlated (r ² = 0.73) with the phosphorus liberated in the first two steps of the Hieltjes and Lijklema extraction by: P = 0.035 (Fe + Al) + 0.001 (P, Fe and Al in mmol g^–1).The iron and aluminum (hydr)oxides are very important fractions in the sediment adsorption capacity for phosphorus. The phosphorus sorption capacity (PSC) is 0.080 mol P (mol (Fe + Al))^–1 and the adsorption constant (k) is 11.9 µmol P l^–1. Here it is assumed that iron and aluminum (hydr)oxide have the same affinity for phosphorus.     

Seven New Mutations in hMSH2, an HNPCC Gene, Identified by Denaturing Gradient-Gel Electrophoresis

Hereditary nonpolyposis colorectal cancer (HNPCC) is a relatively common autosomal dominant cancer-susceptibility condition. The recent isolation of the DNA mismatch repair genes (hMSH2, hMLH1, hPMS1, and hPMS2) responsible for HNPCC has allowed the search for germ-line mutations in affected individuals. In this study we used denaturing gradient-gel electrophoresis to screen for mutations in the hMSH2 gene. Analysis of all the 16 exons of hMSH2, in 34 unrelated HNPCC kindreds, has revealed seven novel pathogenic germ-line mutations resulting in stop codons either directly or through frameshifts. Additionally, nucleotide substitutions giving rise to one missense, two silent, and one useful polymorphism have been identified. The proportion of families in which hMSH2 mutations were found is 21%. Although the spectrum of mutations spread at the hMSH2 gene among HNPCC patients appears extremely heterogeneous, we were not able to establish any correlation between the site of the individual mutations and the corresponding tumor spectrum. Our results indicate that, given the genomic size and organization of the hMSH2 gene and the heterogeneity of its mutation spectrum, a rapid and efficient mutation detection procedure is necessary for routine molecular diagnosis and presymptomatic detection of the disease in a clinical setup.     

Peroxisomes contribute to the acylcarnitine production when the carnitine shuttle is deficient

Fatty acid β-oxidation may occur in both mitochondria and peroxisomes. While peroxisomes oxidize specific carboxylic acids such as very long-chain fatty acids, branched-chain fatty acids, bile acids, and fatty dicarboxylic acids, mitochondria oxidize long-, medium-, and short-chain fatty acids. Oxidation of long-chain substrates requires the carnitine shuttle for mitochondrial access but medium-chain fatty acid oxidation is generally considered carnitine-independent. Using control and carnitine palmitoyltransferase 2 (CPT2)- and carnitine/acylcarnitine translocase (CACT)-deficient human fibroblasts, we investigated the oxidation of lauric acid (C12:0). Measurement of the acylcarnitine profile in the extracellular medium revealed significantly elevated levels of extracellular C10- and C12-carnitine in CPT2- and CACT-deficient fibroblasts. The accumulation of C12-carnitine indicates that lauric acid also uses the carnitine shuttle to access mitochondria. Moreover, the accumulation of extracellular C10-carnitine in CPT2- and CACT-deficient cells suggests an extramitochondrial pathway for the oxidation of lauric acid. Indeed, in the absence of peroxisomes C10-carnitine is not produced, proving that this intermediate is a product of peroxisomal β-oxidation. In conclusion, when the carnitine shuttle is impaired lauric acid is partly oxidized in peroxisomes. This peroxisomal oxidation could be a compensatory mechanism to metabolize straight medium- and long-chain fatty acids, especially in cases of mitochondrial fatty acid β-oxidation deficiency or overload.     

Methanol‐driven enhanced biological phosphorus removal with a syntrophic consortium

The presence of suitable carbon sources for enhanced biological phosphorus removal (EBPR) plays a key role in phosphorus removal from wastewater in urban WWTP. For wastewaters with low volatile fatty acids (VFAs) content, an external carbon addition is necessary. As methanol is the most commonly external carbon source used for denitrification it could be a priori a promising alternative, but previous attempts to use it for EBPR have failed. This study is the first successful report of methanol utilization as external carbon source for EBPR. Since a direct replacement strategy (i.e., supply of methanol as a sole carbon source to a propionic‐fed PAO‐enriched sludge) failed, a novel process was designed and implemented successfully: development of a consortium with anaerobic biomass and polyphosphate accumulating organisms (PAOs). Methanol‐degrading acetogens were (i) selected against other anaerobic methanol degraders from an anaerobic sludge; (ii) subjected to conventional EBPR conditions (anaerobic + aerobic); and (iii) bioaugmented with PAOs. EBPR with methanol as a sole carbon source was sustained in a mid‐term basis with this procedure. Biotechnol. Bioeng. 2013; 110: 391–400. © 2012 Wiley Periodicals, Inc.     

Effect of enrichment on variation and results in the light/dark test

Several confounding factors may influence the outcome of an experiment and the extent of inter-individual variation. The aim of this study was to investigate if cage enrichment induces an effect on experimental mean values and on inter-individual variation in the light/dark paradigm using diazepam as the anxiolytic drug. The behaviour of 216 naive adult male mice of two different strains (BALB/c and C57BL/6) was studied. The animals were housed in groups of four in 'non-enriched', 'enriched' (nesting material) or 'super-enriched' (nest-box, nesting material, wooden gnawing stick and PVC tube) cages. After 5 weeks the animals were assigned to one of three treatments: control (no injection), sham (saline injection i.p.) or diazepam (1 mg/kg bw i.p.) and tested in the light/dark test for 5 min. Variation data were analysed using three different methods (mean absolute deviation, coefficient of variation and power analysis). The C57BL/6 mice scored higher than BALB/c mice in activity related measurements and showed a less 'emotional' behaviour profile in the pharmacological control situation of the light/dark test. In this study the anxiolytic effect of diazepam was clear in BALB/c mice but absent in C57BL/6 mice. Mice housed in enriched and super-enriched cages gained more weight than mice in non-enriched cages, although food intake was not affected. Generally, the strain of mouse had the greatest impact on both mean values and variation. However, there was no consistent increase for one particular strain. The choice of statistical method for analysing variation may influence the interpretation of within-group variability, but none of the methods showed any significant differences between standard and enriched conditions on variability in any of the parameters measured.     

Effects of environmental enrichment for mice: variation in experimental results

This study focused on the effects of different enriched environments for mice in a number of behavioral and physiological parameters in 2 routine laboratory testing procedures: potency testing for tetanus vaccine and stress-induced hyperthermia. The variability in the results was studied by calculating and analyzing mean absolute deviations. Mice from enriched conditions weighed more and consumed more food than mice from standard housing conditions. However, mice from enriched conditions lost more body weight after being housed individually. Other physiological parameters showed no differences. Mice from standard conditions were more active in an open field, suggesting a tendency to overrespond to various stimuli in a testing environment. Mice from enriched environments were more tranquil and easier to handle. The enrichment did not influence the variability in any of the parameters measured, although earlier results and results of other studies suggest that the effects on the variability in results are parameter dependent. When enrichment does not influence variability, there is no reason for not introducing cage enrichment and by doing so contributing to the animals' welfare.     

Project models to improve the ambiance during meal times in Dutch nursing homes: incentives and barriers for implementation

In many Dutch nursing homes, meals are not served in a cozy setting. This study makes an inventory of project models used in nursing homes to improve the ambiance during mealtimes and identifies the beneficial factors (incentives) and barriers of the meal ambiance models. Three different meal ambiance project models were found: restaurant, cooking on the nursing ward and bulkmeals. Each of the three models was experienced as a success by the nursing home inhabitants, the nursing home management and the staff. They pointed out that the cooperation and motivation of staff, sufficient space and management support were the most important beneficial factors of the project models. The most important barriers were absence of cooperation and motivation of the staff, insufficient finances and insufficient personnel.