PURIFICATION OF PHOSPHATE-DEPENDENT PIG BRAIN GLUTAMINASE

Abstract— A procedure for preparing highly purified phosphate-activated glutaminase (EC 3.5.1.2, L-glutamine amidohydrolase) from pig brain is described. The main steps consist of extraction with acetone, followed by sodium sulphate fractionation of the solubilized acetone powder. Thereafter, solubilization by dialysis against a buffer containing tris-HC1, mercaptoethanol, and EDTA, followed by precipitation with phosphate-borate, is repeated twice. The final preparation contains no impurities which can be detected by polyacrylamide gel electrophoresis, isoelectric focusing, and sedimentation equilibrium centrifugation. By the latter method, molecular weight is determined to be 187,000. By polyacrylamide gel electrophoresis in sodium dodecyl sulphate, one protein band with molecular weight 64,000 is found.     

Rapid quantitative assay for acaricidal effects on Sarcoptes scabiei var. suis and Otodectes cynotis

Brimer et al. (Vet. Parasitol. 51: 123-135, 1993 and 59: 249-255, 1995) developed a migration assay for acaricidal effect of acetylcholinesterase inhibitors and macrocyclic lactones utilising Sarcoptes scabiei var. suis mites. In contrast to many others, this assay is fully quantitative but quite time-consuming. The aim of the present investigation was to modify this assay to become faster and simpler. As a result accurate determinations can now be obtained within 6h, as opposed to 24h. Furthermore it was demonstrated that also Otodectes cynotis mites can be used with only minor modifications of the procedures. The cholinesterase inhibitor diazinon and the formamide amitraz were used as acaricides. Thus, the mite migration assay now has been proven useful for acaricidal compounds belonging to three chemical groups with different modes of action, namely organophosphorous cholinesterase inhibitors, macrocyclic lactones acting on the glutamanergic/GABAegic motoneurons, and formamide inhibitors of the octopamine systems of arthropods.     

Length-independent structural similarities enrich the antibody CDR canonical class model

Complementarity-determining regions (CDRs) are antibody loops that make up the antigen binding site. Here, we show that all CDR types have structurally similar loops of different lengths. Based on these findings, we created length-independent canonical classes for the non-H3 CDRs. Our length variable structural clusters show strong sequence patterns suggesting either that they evolved from the same original structure or result from some form of convergence. We find that our length-independent method not only clusters a larger number of CDRs, but also predicts canonical class from sequence better than the standard length-dependent approach.

To demonstrate the usefulness of our findings, we predicted cluster membership of CDR-L3 sequences from 3 next-generation sequencing datasets of the antibody repertoire (over 1,000,000 sequences). Using the length-independent clusters, we can structurally classify an additional 135,000 sequences, which represents a ～20% improvement over the standard approach. This suggests that our length-independent canonical classes might be a highly prevalent feature of antibody space, and could substantially improve our ability to accurately predict the structure of novel CDRs identified by next-generation sequencing.     

A Study on the Effects of Tactical Drenchings with a Benzimidazole against Ostertagiasis in Calves

In the present study tactical fenbendazole treatments against ostertagiasis in calves were given at short intervals towards the end of the grazing season. This prevented clinical disease and had moderate effects on parasitic loads in animals that stayed on the same pasture throughout the season. The feasibility of tactical treatments is discussed in relation to various strategical control measures.     

Benthic decomposition of Ulva lactuca: A controlled laboratory experiment

The degradation of an Ulva lactuca mat (0.2 kg dw m⁻²) was studied in a controlled flow-through mesocosm for 31 d. Sediment chambers without U. lactuca served as controls. Fluxes of ∑CO₂, O₂, inorganic nitrogen, and urea were determined during the incubation period in addition to sulfate reduction rates, POC and PON content, enumeration of specific bacterial populations and evaluation of the physiological state of the added U. lactuca thalli. After U. lactuca addition to the chambers, there was an immediate increase in the efflux of ∑CO₂ from 11 to 27 mmol-C m⁻² d⁻¹ and a concomitant increase in O₂ uptake from 11 to 23 mmol m⁻² d⁻¹. These effluxes remained elevated throughout the incubation period. In contrast, the NH₄⁺ efflux increased from 0.1 to 1.8 mmol NH₄⁺ m⁻² d⁻¹ during the first 3 d of incubation, followed by 6 d with a constant efflux rate, after which time it decreased gradually to 0.3 mmol NH₄⁺ m⁻² d⁻¹ by the end of the experiment. In total, NH₄⁺accounted for 83% of the total nitrogen efflux after addition of U. lactuca. During the 31 d incubation period there was a continuous colonization of the thalli by bacteria. Sulfate reducers associated with the thalli accounted for 3% of the carbon oxidation on day 31. The molar C:N ratio in mineralization products (the ratio between the efflux of ∑CO₂ and NH₄⁺ + NO₂⁻ + NO₃⁻) increased from 15 mol mol⁻¹ at day 11 after U. lactuca addition to >80 mol mol⁻¹ by the end of the incubation. Since the C:N ratio in the mineralization products was much higher than the original thallus material (8.9 mol mol⁻¹) it is probable that a preferential incorporation of NH₄⁺ into the increasing bacterial biomass occurred. The nitrogen for bacterial growth was most likely obtained from degradation of U. lactuca thalli as there was no stimulation of urea-N turnover in the sediment during incubation. The net increase in bacteria cell number in the 18-mm thick thallus layer was estimated to be 7.6 × 10⁹ to 2.4 × 10¹⁰ bacterial cells cm⁻³. In contrast, the bacterial cell number remained constant in the −Ulva incubations.     

The contribution of CLIP2 haploinsufficiency to the clinical manifestations of the Williams-Beuren syndrome

Williams-Beuren syndrome is a rare contiguous gene syndrome, characterized by intellectual disability, facial dysmorphisms, connective-tissue abnormalities, cardiac defects, structural brain abnormalities, and transient infantile hypercalcemia. Genes lying telomeric to RFC2, including CLIP2, GTF2I and GTF2IRD1, are currently thought to be the most likely major contributors to the typical Williams syndrome cognitive profile, characterized by a better-than-expected auditory rote-memory ability, a relative sparing of language capabilities, and a severe visual-spatial constructive impairment. Atypical deletions in the region have helped to establish genotype-phenotype correlations. So far, however, hardly any deletions affecting only a single gene in the disease region have been described. We present here two healthy siblings with a pure, hemizygous deletion of CLIP2. A putative role in the cognitive and behavioral abnormalities seen in Williams-Beuren patients has been suggested for this gene on the basis of observations in a knock-out mouse model. The presented siblings did not show any of the clinical features associated with the syndrome. Cognitive testing showed an average IQ for both and no indication of the Williams syndrome cognitive profile. This shows that CLIP2 haploinsufficiency by itself does not lead to the physical or cognitive characteristics of the Williams-Beuren syndrome, nor does it lead to the Williams syndrome cognitive profile. Although contribution of CLIP2 to the phenotype cannot be excluded when it is deleted in combination with other genes, our results support the hypothesis that GTF2IRD1 and GTF2I are the main genes causing the cognitive defects associated with Williams-Beuren syndrome.     

Genetic evidence for population expansion in Hydrotaea irritans (Fallèn) (Diptera: Muscidae)

Allozyme variation in the sheep headfly Hydrotaea irritans was studied on two spatial scales. Geographic variation among seven Danish and one Dutch population revealed significant but rather low genetic differentiation with F _ST = 0.01 over all loci. The Dutch population was on average not more different from the Danish populations than the Danish populations from each other. Allele frequencies were very skewed with the most common allele always exceeding 0.85 and usually 0.9 in frequency, but with many rare alleles at some loci. Tests for neutrality of the variation at the nine polymorphic loci revealed highly significant deviations from expected homozygosity in this species, which was not found in a comparative analysis of allozyme variation at similar loci of seven other Hydrotaea species. To explain the peculiar observed pattern of allozyme variation in H. irritans , it is suggested that this species has successfully expanded its range and spread through northern and central Europe in the recent past. Alternatively, H. irritans may have recently invaded a new niche, resulting in increased abundance of the species and subsequent dispersal to former areas of the species distribution.     

Genome-wide copy number profiling of single cells in S-phase reveals DNA-replication domains

Single-cell genomics is revolutionizing basic genome research and clinical genetic diagnosis. However, none of the current research or clinical methods for single-cell analysis distinguishes between the analysis of a cell in G1-, S- or G2/M-phase of the cell cycle. Here, we demonstrate by means of array comparative genomic hybridization that charting the DNA copy number landscape of a cell in S-phase requires conceptually different approaches to that of a cell in G1- or G2/M-phase. Remarkably, despite single-cell whole-genome amplification artifacts, the log2 intensity ratios of single S-phase cells oscillate according to early and late replication domains, which in turn leads to the detection of significantly more DNA imbalances when compared with a cell in G1- or G2/M-phase. Although these DNA imbalances may, on the one hand, be falsely interpreted as genuine structural aberrations in the S-phase cell’s copy number profile and hence lead to misdiagnosis, on the other hand, the ability to detect replication domains genome wide in one cell has important applications in DNA-replication research. Genome-wide cell-type-specific early and late replicating domains have been identified by analyses of DNA from populations of cells, but cell-to-cell differences in DNA replication may be important in genome stability, disease aetiology and various other cellular processes.