Inhibition of Real-Time PCR in DNA Extracts from Aquifer Sediment

Variation in inhibition of real-time PCR was investigated with DNA extracts from 50 aquifer sediment core samples of 5 cm length collected through a 2.5 meter vertical profile across a landfill leachate plume. The inhibition was quantified using an internal control of the green fluorescent protein ( gfp ) gene, which was spiked into the real-time PCR reactions. The inhibition was investigated at two gfp gene concentrations: at 1.7 · 10 ⁷ gfp gene copies/g sediment (5.1 · 10 ⁴ gfp gene copies/PCR reaction) and at 1.7 · 10 ⁵ gfp gene copies/g sediment (5.1 · 10 ² gfp gene copies/PCR reaction). Despite the low TOC content of the sediment (average 0.4 mg C/g dw) the average real-time PCR response was partially inhibited, compared to a reference (pure water), at both high and low gfp concentrations. The relative amplification (reference = 1) was 0.85 ± 0.20 (high) and 0.66 ± 0.23 (low), showing significantly (P < 0.05) stronger inhibition at the lower target gene concentration. The inhibition of the real-time PCR did not show a systematic variation in the vertical profile related to plume position but variations were significant on a small scale of 5–15 cm depth intervals. One of the 50 samples failed to produce a signal with either concentration of the gfp internal control and three other samples inhibited real-time PCR at both high and low gfp concentration. These 4 samples, which were the samples with the highest inhibition, were the only DNA extracts with a visible brown colouration, indicating contents of humic-like substances. Elevated absorbance at 400 nm of these samples also indicated that humic-like substances were responsible for inhibition. However, other factors not associated with either absorbance or TOC may have contributed to the inhibition in less inhibited samples since the variation in real-time PCR response could not be sufficiently explained by absorbance or TOC. The results of this study suggest that an internal control is needed in real-time PCR reactions with DNA from environmental samples due to variation in inhibition to correctly quantify the number of target genes, especially at low target gene concentrations, when dilution of DNA extracts is not practical.     

Data standards, sense and stability: Scratchpads, the ICZN and ZooBank

The International Commission of Zoological Nomenclature has used the Scratchpads platform (currently being developed and maintained by ViBRANT) as the foundation for its redesigned website and as a platform for engaging with its users. The existing Scratchpad tools, with extensions to provide additional functions, have allowed for a major transformation in presentation of linked nomenclatural tools. Continued development of the new website will act as a springboard for the ICZN to participate more fully in the wider community of biodiversity informatics.     

Genotype, enzyme activity, glutathione level, and clinical phenotype in patients with glutathione synthetase deficiency

Glutathione synthetase (GS) deficiency is a rare autosomal recessive disorder. The clinical phenotype varies widely, and nearly 30 different mutations in the GSS gene have been identified. In the present study, genotype, enzyme activity, metabolite levels and clinical phenotype were evaluated in 41 patients from 33 families. From some of the patients, data on glutathione (GSH) levels and -glutamylcysteine levels in cultured fibroblasts were also available. Twenty-seven different mutations were found: 14 missense, 9 splice, 2 deletions, 1 insertion and 1 nonsense mutation. Twenty-three patients were homozygous and 18 were compound heterozygous. The moderate and severe clinical phenotypes could not be distinguished based on enzyme activity, GSH or -glutamylcysteine levels in cultured fibroblasts. However, in fibroblasts, the residual GS activity was correlated with the GSH level. All mutations causing frameshifts, premature stop codons or aberrant splicing were associated with moderate or severe clinical phenotypes including haemolytic anaemia, 5-oxoprolinuria, and (in several forms) neurodevelopmental signs. The data indicate that additional genetic or environmental factors modify at least the moderate and severe phenotypes and that the clinical classification given to the patients may be influenced by variation in follow-up. The type of mutation involved can, to some extent, predict a mild versus a more severe phenotype.Electronic Supplementary Material Suplementary material is available for this article at .Electronic database information: Online Mendelian Inheritance in Man, (MIM 266130 for GS deficiency); GenBank, ( nos. AL133324.13 for genomic DNA and NM_000178.2 for mRNA were used for sequence referencing)     

Neutrophil-derived heparin-binding protein (HBP/CAP37) deposited on endothelium enhances monocyte arrest under flow conditions

In acute inflammation, infiltration of neutrophils often precedes a second phase of monocyte invasion, and data in the literature suggest that neutrophils may directly stimulate mobilization of monocytes via neutrophil granule proteins. In this study, we present a role for neutrophil-derived heparin-binding protein (HBP) in monocyte arrest on endothelium. Adhesion of neutrophils to bovine aorta endothelial cells (ECs) or HUVEC-triggered secretion of HBP and binding of the protein to the EC surface. Blockade of neutrophil adhesion by treatment with a mAb to CD18 greatly reduced accumulation of HBP. In a flow chamber model, immobilized recombinant HBP induced arrest of human monocytes or monocytic Mono Mac 6 (MM6) cells to activated EC or plates coated with recombinant adhesion molecules (E-selectin, P-selectin, VCAM-1). However, immobilized recombinant HBP did not influence arrest of neutrophils or lymphocytes. Treatment of MM6 cells with recombinant HBP evoked a rapid and clear-cut increase in cytosolic free Ca(2+) that was found to be critical for the HBP-induced monocyte arrest inasmuch as pretreatment with the intracellular calcium chelating agent BAPTA-AM abolished the evoked increase in adhesion. Thus, secretion of a neutrophil granule protein, accumulating on the EC surface and promoting arrest of monocytes, could contribute to the recruitment of monocytes at inflammatory loci.     

Neutrophil elastase sorting involves plasma membrane trafficking requiring the C-terminal propeptide

The primary granules/secretory lysosomes of neutrophils store mature neutrophil elastase (NE) as a luminal protein after proteolytic removal of N-terminal and C-terminal pro-peptides from a proform of NE. The N-terminal pro-peptide prevents premature activation that might be toxic to the cell, but the C-terminal pro-peptide has no defined function. In this study, we investigated the role of the C-terminal pro-peptide in trafficking of NE by expressing, in rat basophilic leukemia (RBL) cells, both wild-type NE and the mutant NE/Delta248-267, which lacks the C-terminal pro-peptide. Both transfected proteins were found to be targeted to secretory lysosomes. In addition, results from antibody ligation and cell-surface biotinylation indicated that proform of NE was targeted to the plasma membrane, and then subjected to endocytosis. The results were supported by the detection of targeting of the proform to the plasma membrane followed by internalization both in RBL cells and normal granulopoietic precursor cells. Targeting of NE to the plasma membrane required the C-terminal pro-peptide as NE/Delta248-267 expressed in RBL cells bypassed plasma membrane trafficking. Our results indicate targeting of a population of NE to the plasma membrane and internalization dependent on the C-terminal NE pro-peptide.     

Spirochetes in gastropods from Lake Baikal and North American freshwaters: new multi-family, multi-habitat host records

We describe the first records of spirochetes in the gut of fourteen species of continental gastropods from a range of habitats and representing six families (Amnicolidae, Baicaliidae, Bithyniidae, Pyrgulidae, Lithoglyphidae and Benedictiidae). The bacteria were mainly found in the crystalline style sac, as has been reported in marine bivalves. The surveyed habitats include water bodies in North America and Eurasia, including deep water hydrothermal vent and gas hydrate zones in Lake Baikal. Spirochetes were present both in mature and young snails, but were not detected in embryos before hatching, indicating lateral transfer. The surveyed gastropods range in trophic strategy, including phyto-, detrito- and bacteriophagous grazers and filter feeders. Our results indicate that spirochetes are commensal in the surveyed gastropods with potential limited benefit and no detriment to the host animal. We suggest that the specialized internal habitat of the crystalline style sac in molluscs is likely to reveal unrecognized spirochete diversity that will shed new light on gastropod trophic ecology and spirochete diversity.     

gC1qR expression in normal and pathologic human tissues: differential expression in tissues of epithelial and mesenchymal origin

The gC1qR (i.e., gC1q receptor, gC1q binding protein, p32, p33) is a multifunctional cellular protein that interacts with components of the complement, kinin, and coagulation cascades and select microbial pathogens. Enhanced gC1qR expression has been reported in adenocarcinomas arising in a variety of organs. The present study compared gC1qR expression in normal, inflammatory, dysplastic, and malignant tissue of epithelial and mesenchymal origin. gC1qR expression was visualized in tissue sections by immunohistochemistry using the 60.11 monoclonal antibody (i.e., IgG(1) mouse monoclonal antibody directed against gC1qR) and the UltraVision LP Detection System. Sections were counterstained with hematoxylin and examined by light microscopy. Strongest gC1qR expression was noted in epithelial tumors of breast, prostate, liver, lung, and colon, as well as in squamous and basal cell carcinoma of the skin. However, increased gC1qR staining was appreciated also in inflammatory and proliferative lesions of the same cell types, as well as in normal continuously dividing cells. In contrast, tumors of mesenchymal origin generally stained weakly, with the exception of osteoblasts, which stained in both benign and malignant tissues. The data suggest that increased gC1qR expression may be a marker of benign and pathologic cell proliferation, particularly in cells of epithelial origin, with potential diagnostic and therapeutic applications.     

Trimming down a protein structure to its bare foldons: spatial organization of the cooperative unit

Folding of the ribosomal protein S6 is a malleable process controlled by two competing, and partly overlapping, folding nuclei. Together, these nuclei extend over most of the S6 structure, except the edge strand β2, which is consistently missing in the folding transition states; despite being part of the S6 four-stranded sheet, β2 seems not to be part of the cooperative unit of the protein. The question is then whether β2 can be removed from the S6 structure without compromising folding cooperativity or native state integrity. To investigate this, we constructed a truncated variant of S6 lacking β2, reducing the size of the protein from 96 to 76 residues (S6(Δβ2)). The new S6 variant expresses well in Escherichia coli and has a well dispersed heteronuclear single quantum correlation spectrum and a perfectly wild-type-like crystal structure, but with a smaller three-stranded β-sheet. Moreover, S6(Δβ2) displays an archetypical v-shaped chevron plot with decreased slope of the unfolding limb, as expected from a protein with maintained folding cooperativity and reduced size. The results support the notion that foldons, as defined by the structural distribution of the folding nuclei, represent a property-based level of hierarchy in the build-up of larger protein structures and suggest that the role of β2 in S6 is mainly in intermolecular binding, consistent with the position of this strand in the ribosomal assembly.