Quality control in the determination of cortisol in plasma/serum by using, on every sample, two different three-step separation methods including ultrafiltration, restricted-access high-performance liquid chromatography and reversed-phase high-performance liquid chromatography, and contrasting results to immunoassays

Tests of HPLC columns with restricted access, polymer covered alumina, polymer, and different ODS phases showed that base-acid compatible ODS columns gave the best peak shapes of cortisol, internal standard, as well as of plasma/serum (P/S) matrix components. Further trials with cortisol in P/S showed that three separation steps were essential in order to obtain chromatographic data which were superior to immunoassay data. Also, sufficient confidence in results required determination of each sample with two newly developed separation methods: (a) pre-separation with a restricted access column, concentration of the desired cut with a 20 mm base-acid compatible ODS column, and analysis with a 250 mm column filled with the same ODS; (b) pre-separation with an ultrafilter followed by the last two steps in (a). For detection UV was preferred over fluorescence. This twin multistep chromatography showed that immunoassays were very treacherous in that they produced a spectrum of results ranging from good to untenable without any warning whatever about functionality. The measurement of official controls, with reference values derived via gas chromatography-isotope dilution mass spectrometry, also demonstrated the superiority of the double HPLC method.     

Detection of Plasmodium falciparum DNA in a microtitration plate-based hybridization test

A rapid DNA-test, depending on the affinity based hybrid collection principle, was developed for the detection of Plasmodium falciparum DNA from clinical specimens. In this method, hybridization takes place in solution and the hybrids are collected onto a solid phase for measurement. Two probes are used, one labelled with an affinity tag (biotin) and the other with a detectable label (32P). In the present test a single oligonucleotide complementary to a 21-base pair sequence which is highly repeated in the parasite genome served both as capture and detector probe. The test is a 2-h hybridization performed in streptavidin coated microtitration plate wells, onto which the labelled hybrids simultaneously bind. The sensitivity of the assay with a crude erythrocyte lysate specimen was 1.6 x 10(9) repeat units corresponding to about 160 parasites in one microliter blood. The results allowed quantification of the repeat sequences and thus estimation of the degree of parasitemia in clinical specimens.     

Affect and Cognition in Attitude Formation toward Familiar and Unfamiliar Attitude Objects

At large attitudes are built on earlier experience with the attitude object. If earlier experiences are not available, as is the case for unfamiliar attitude objects such as new technologies, no stored evaluations exist. Yet, people are still somehow able to construct attitudes on the spot. Depending on the familiarity of the attitude object, attitudes may find their basis more in affect or cognition. The current paper investigates differences in reliance on affect or cognition in attitude formation toward familiar and unfamiliar realistic attitude objects. In addition, individual differences in reliance on affect (high faith in intuition) or cognition (high need for cognition) are taken into account. In an experimental survey among Dutch consumers (N = 1870), we show that, for unfamiliar realistic attitude objects, people rely more on affect than cognition. For familiar attitude objects where both affective and cognitive evaluations are available, high need for cognition leads to more reliance on cognition, and high faith in intuition leads to more reliance on affect, reflecting the influence of individually preferred thinking style. For people with high need for cognition, cognition has a higher influence on overall attitude for both familiar and unfamiliar realistic attitude objects. On the other hand, affect is important for people with high faith in intuition for both familiar and unfamiliar attitude objects and for people with low faith in intuition for unfamiliar attitude objects; this shows that preferred thinking style is less influential for unfamiliar objects. By comparing attitude formation for familiar and unfamiliar realistic attitude objects, this research contributes to understanding situations in which affect or cognition is the better predictor of overall attitudes.     

Stop and Go – Waves of Tarsier Dispersal Mirror the Genesis of Sulawesi Island

The Indonesian island of Sulawesi harbors a highly endemic and diverse fauna sparking fascination since long before Wallace’s contemplation of biogeographical patterns in the region. Allopatric diversification driven by geological or climatic processes has been identified as the main mechanism shaping present faunal distribution on the island. There is both consensus and conflict among range patterns of terrestrial species pointing to the different effects of vicariant events on once co-distributed taxa. Tarsiers, small nocturnal primates with possible evidence of an Eocene fossil record on the Asian mainland, are at present exclusively found in insular Southeast Asia. Sulawesi is hotspot of tarsier diversity, whereby island colonization and subsequent radiation of this old endemic primate lineage remained largely enigmatic. To resolve the phylogeographic history of Sulawesi tarsiers we analyzed an island-wide sample for a set of five approved autosomal phylogenetic markers (ABCA1, ADORA3, AXIN1, RAG1, and TTR) and the paternally inherited SRY gene. We constructed ML and Bayesian phylogenetic trees and estimated divergence times between tarsier populations. We found that their arrival at the Proto-Sulawesi archipelago coincided with initial Miocene tectonic uplift and hypothesize that tarsiers dispersed over the region in distinct waves. Intra-island diversification was spurred by land emergence and a rapid succession of glacial cycles during the Plio-Pleistocene. Some tarsier range boundaries concur with spatial limits in other taxa backing the notion of centers of faunal endemism on Sulawesi. This congruence, however, has partially been superimposed by taxon-specific dispersal patterns.     

Mutations of the CD40 ligand gene in 13 Japanese patients with X-linked hyper-IgM syndrome

X-linked hyper-IgM syndrome (XHIM) is a rare primary immunodeficiency caused by a defective CD40 ligand. We identified mutations of the CD40 ligand gene in 13 unrelated Japanese XHIM patients. Of the four patients with missense mutations, one had a mutation within the transmembrane domain, and the three others had mutations affecting the TNF homology region of the extracellular domain. Two of the missense mutations resulted in the substitution of amino acids that are highly conserved in TNF family proteins. Three patients had nonsense mutations, all of which resulted in the truncation of the TNF homology domain of the CD40 ligand. Three patients had genomic DNA deletions of 2, 3 or 4 nucleotides, respectively. All of the deletions were flanked by direct repeat sequences, suggesting that these deletions were caused by slipped mispairing. Three patients had mutations within introns resulting in altered splicing, and multiple splicing products were found in one patient. Thus, each of the 13 Japanese patients had different mutations, 9 of them being novel mutations. These results indicate that mutations in XHIM are highly heterogeneous, although codon 140 seems to be a hot spot of the CD40 ligand gene since two additional point mutations were located at Trp 140, bringing the total numbers of mutations affecting codon 140 to six. In one XHIM family with a missense mutation, prenatal diagnosis was performed by single-strand conformation polymorphism analysis of genomic DNA of a male fetus. Received: 20 August 1996     

The regulatory strength: How to be precise about regulation and homeostasis

The concepts of regulation and homeostasis are of frequent use but lack a single universally accepted definition. Here we propose a definition of theregulatory strength andhomeostatic strength, which allow to assess the importance of a regulatory pathway in a quantitative fashion.     

Characterization of newly established colorectal cancer cell lines: correlation between cytogenetic abnormalities and allelic deletions associated with multistep tumorigenesis

We have established a series of 20 colorectal cancer cell lines and performed cytogenetic and RFLP analyses to show that the recurrent genetic abnormalities of chromosomes 1, 5, 17 and 18 associated with multistep tumorigenesis in colorectal cancer, and frequently detected as recurrent abnormalities in primary tumours, are also retained in long-term established cell lines. Earlier studies by us and other investigators showed that allelic losses of chromosomes 1 and 17 in primary colorectal cancers predicted poorer survival for the patients (P = 0.03). We utilized the cell lines to identify specific chromosomal sites or gene(s) on chromosomes 1 and 17 which confer more aggressive phenotype. Cytogenetic deletions of chromosome 1p were detected in 14 out of the 20 (70%) cell lines, whereas allelic deletions for 1p using polymorphic markers were detected in 13 out of 18 (72%) informative cell lines for at least one polymorphic marker. We have performed Northern blotting, immunohistochemical staining (p53 mRNA, protein) and RFLP analysis using several probes including p53 and nm23. RFLP analysis using a total of seven polymorphic markers located on 17p and 17q arms showed allelic losses aroundthe p53 locus in 16 out of the 20 cell lines (80%), four of which were losses of thep53 locus itself. In addition, seven cell lines (out of nine informative cases) also showed losses of thenm23 gene, four with concurrent losses of thep53 locus, while the remaining three were homozygous. In addition, five out of seven cell lines withnm23 deletions were derived from hepatic metastatic tumours, and one cell line was obtained from recurrent tumour. A comparison between allelic deletions of 1p and functional loss ofnm23 gene revealed a close association between these two events in cell lines derived from hepatic metastasis. Following immunohistochemical staining, nine out of the twenty cell lines showed high levels (25–80%) of mutant p53, four showed intermediate levels (>20%), and seven had undetectable levels of the protein. Of these seven, four showed complete absence of mRNA. Of the remaining three cell lines one showed aberrant mRNA due to germline rearrangement of thep53 gene, whereas in two cell lines normal levels of mRNA were present. Nineteen of the 20 cell lines had normal germline configurations for thep53 gene, while one showed a rearrangement. These data suggest that functional loss ofp53 andnm23 genes accomplished by a variety of mechanisms may be associated with poor prognosis and survival. In addition, concurrent deletions of chromosome regions 17p, 17q and 1p were closely associated with high-stage hepatic metastatic disease. These cell lines with well-characterized genetic alterations and known clinical history provide an invaluable source of material for various biological and clinical studies relating to multistep colorectal tumorigenesis.     

Prevention and treatment of tail biting in weaned piglets

The aims of this study were to evaluate four preventive measures and two curative treatments of tail biting. The preventive measures were: chain, rubber hose, straw rack (5 g/pig/day) and the provision of straw on the floor twice daily by hand (2 × 10 g/pig/day). The two curative treatments, which were applied following the onset of tail biting in a pen were: straw twice daily (as in the fourth preventive measure) and the removal of the biter. In total, 960 undocked weaned piglets (10 piglets per pen) were observed during 5 weeks. Tail lesions (none, bite marks and wounds) were recorded daily. The incidence of pens with wounded pig tails was significantly lower when straw was provided twice daily (8% of pens) compared to the chain (58% of pens) and rubber hose (54% of pens) treatment, but did not differ significantly from the straw rack treatment (29% of pens). Tails with bite marks were significantly less common in pens with twice daily straw (16% of pens) compared to chain (88% of pens), rubber hose (79% of pens) and straw rack (75% of pens). No significant difference was found between the curative treatments. Both treatments showed a reduced incidence of red fresh blood on the tails at days 1–9 following curative treatment, compared to day 0. However, neither curative treatment eliminated tail biting entirely. In conclusion, this study indicates that tail biting is best prevented with a small amount of straw, provided twice daily, and to a lesser extent with a straw rack, compared to providing a chain or a rubber hose. Once tail biting has occurred, providing a small amount of straw twice daily and removing the biter appears to be equally effective.     

Comparison of Different Methods for the Evaluation of Treatment Effects from the Sleep EEG of Patients with Major Depression

In healthy subjects, sleep has a typical structure of three to five cyclic transitions between different sleep states. In major depression, this regular pattern is often destroyed but can be reestablished during successful treatment. The differences between healthy and abnormal sleep are generally assessed in a time-consuming process, which consists of determining the nightly variations of the sleep states (the hypnogram) based on visual inspection of the electroencephalogram (EEG), electrooculogram, and electromyogram. In this study, three different methods of sleep EEG analysis (spectrum, outlier, and recurrence analysis) have been examined with regard to their ability to extract information about treatment effects in patients with major depression. Our data suggest that improved sleep patterns during treatment with antidepressant medication can be identified with an appropriate analysis of the EEG. By comparing different methods, we have found that many treatment effects identified by spectrum analysis can be reproduced by the much simpler technique of outlier analysis. Finally, the cyclic structure of sleep and its modification by antidepressant treatment is best illustrated by a non-linear approach, the so-called recurrence method.