Apolipoprotein E polymorphism in the Netherlands and its effect on plasma lipid and apolipoprotein levels

Summary By isoelectric focusing of delipidated sera followed by immunoblotting we studied the apolipoprotein (apo) E polymorphism in 2018 randomly selected 35-years-old males from three different areas in the Netherlands. Comparison of the APOE allele (E^*2, E^*3, and E^*4) frequencies estimated in this study with those reported for several other population samples showed that there are marked differences between the Dutch population and the populations of Japan, New Zealand, Finland, and the United States. These differences in APOE allele frequencies appeared to be mainly due to differences in frequencies of the E^*2 allele (decreased in Japan and Finland; increased in New Zealand) and the E^*4 allele (increased in Finland; decreased in Japan and the United States). No difference in APOE allele frequencies was found between the Dutch population and the populations of West Germany and Scotland. Measurements of plasma cholesterol and apo B and E concentrations showed that the E^*4 allele is associated with elevated plasma cholesterol and apo B levels and with decreased apo E concentrations, whereas the opposite is true for the E^*2 allele. In the Dutch population, the sum of average allelic effects of the common APOE alleles on plasma cholesterol and apo B levels is 6.8% and 14.2%, respectively, of the total population mean. The total average allelic effect on plasma apo E concentrations was more pronounced (50.1%), suggesting that the APOE alleles primarily affect apo E concentrations rather than plasma cholesterol and apo B levels. This hypothesis is sustained by the observation that for plasma apo E levels the genetic variance associated with the APOE gene locus contributed about 18% to the total phenotypic variance. For plasma cholesterol and apo B this contribution was only 1.4% and 2.3% and is relatively low as compared with that reported for other population samples.     

Adenosine kinase deficiency disrupts the methionine cycle and causes hypermethioninemia, encephalopathy, and abnormal liver function

Four inborn errors of metabolism (IEMs) are known to cause hypermethioninemia by directly interfering with the methionine cycle. Hypermethioninemia is occasionally discovered incidentally, but it is often disregarded as an unspecific finding, particularly if liver disease is involved. In many individuals the hypermethioninemia resolves without further deterioration, but it can also represent an early sign of a severe, progressive neurodevelopmental disorder. Further investigation of unclear hypermethioninemia is therefore important. We studied two siblings affected by severe developmental delay and liver dysfunction. Biochemical analysis revealed increased plasma levels of methionine, S-adenosylmethionine (AdoMet), and S-adenosylhomocysteine (AdoHcy) but normal or mildly elevated homocysteine (Hcy) levels, indicating a block in the methionine cycle. We excluded S-adenosylhomocysteine hydrolase (SAHH) deficiency, which causes a similar biochemical phenotype, by using genetic and biochemical techniques and hypothesized that there was a functional block in the SAHH enzyme as a result of a recessive mutation in a different gene. Using exome sequencing, we identified a homozygous c.902C>A (p.Ala301Glu) missense mutation in the adenosine kinase gene (ADK), the function of which fits perfectly with this hypothesis. Increased urinary adenosine excretion confirmed ADK deficiency in the siblings. Four additional individuals from two unrelated families with a similar presentation were identified and shown to have a homozygous c.653A>C (p.Asp218Ala) and c.38G>A (p.Gly13Glu) mutation, respectively, in the same gene. All three missense mutations were deleterious, as shown by activity measurements on recombinant enzymes. ADK deficiency is a previously undescribed, severe IEM shedding light on a functional link between the methionine cycle and adenosine metabolism.     

Structural insights into cognate versus near-cognate discrimination during decoding

The structural basis of the tRNA selection process is investigated by cryo-electron microscopy of ribosomes programmed with UGA codons and incubated with ternary complex (TC) containing the near-cognate Trp-tRNA(Trp) in the presence of kirromycin. Going through more than 350 000 images and employing image classification procedures, we find ～8% in which the TC is bound to the ribosome. The reconstructed 3D map provides a means to characterize the arrangement of the near-cognate aa-tRNA with respect to elongation factor Tu (EF-Tu) and the ribosome, as well as the domain movements of the ribosome. One of the interesting findings is that near-cognate tRNA's acceptor stem region is flexible and CCA end becomes disordered. The data bring direct structural insights into the induced-fit mechanism of decoding by the ribosome, as the analysis of the interactions between small and large ribosomal subunit, aa-tRNA and EF-Tu and comparison with the cognate case (UGG codon) offers clues on how the conformational signals conveyed to the GTPase differ in the two cases.     

Daunorubicin does not induce immunohistochemically detectable endothelial dysfunction in rabbit aorta and femoral artery

Anthracyclines are one of the most effective anticancer drugs ever developed, but their clinical use has been hampered by the risk of severe cardiotoxicity. In this study, we investigated whether rabbits exposed to a different cumulative dose of anthracycline suffer from immunohistochemically detectable vascular toxicity and endothelial dysfunction. Daunorubicin (3 mg/kg, i.e. 50 mg/m2) was administered i.v. to rabbits once weekly for 1-10 weeks to reach different cumulative doses of the drug (50-500 mg/m2), while control rabbits received saline. The rabbits were sacrificed either 24 hours or 7 days after reaching each particular cumulative dose, and aortas and right femoral arteries were collected for immunohistochemical analysis. Immunohistochemical analysis showed ICAM-1 staining in many aortas from both saline and daunorubicin-treated rabbits without any relationship to the anthracycline treatment. On the contrary, unlike in the lipopolysaccharide-treated or hypercholesterolemic rabbits, no distinct immunoreactivity for other markers of inflammation, oxidative and nitrosative stress (VCAM-1, 4-HNE, iNOS and nitrotyrosine) were detected in aortas and femoral arteries from either control or daunorubicin-treated animals. No relationship to the cumulative dose of the drug or post-expose set up of harvesting was found. In this study, we have demonstrated that daunorubicin does not induce gross histopathological changes in the studied arteries and it fails to induce immunohistochemically detectable endothelial dysfunction. Thus, we propose that endothelial cells are much less susceptible to anthracycline toxicity than cardiac myocytes. In addition, our data suggest that vascular toxicity of anthracyclines plays rather a minor role in the cardiovascular complications of anthracycline chemotherapy.     

Comparative study of MLPA-FISH to determine DNA copy number alterations in neuroblastic tumors

Neuroblastoma tumor cells show complex combinations of genetic aberrations, and to date many different methods have been used for their detection. To apply genome-wide techniques, such as Multiplex Ligation-dependent Probe Amplification (MLPA), in routine diagnosis their validation is appropriate and necessary. DNA copy number alterations in 129 cases of neuroblastic tumors were detected using MPLA, and the results validated by Fluorescence In Situ Hybridization (FISH) (MYCN gene, 1p36, 11q and 17q). Kappa index values showed very good concordance between the two techniques in detecting homogeneous MYCN amplification (1); 11q deletion (0.908) and 17q gain (0.922). The validation results showed that MLPA is a highly efficient technique for diagnosis based on the genetic aberrations in relevant regions in neuroblastoma, showing a high concordance with FISH.     

Comparison of dysferlin expression in human skeletal muscle with that in monocytes for the diagnosis of dysferlin myopathy

Background

Dysferlinopathies are caused by mutations in the dysferlin gene (DYSF). Diagnosis is complex due to the high clinical variability of the disease and because dysferlin expression in the muscle biopsy may be secondarily reduced due to a primary defect in some other gene. Dysferlin is also expressed in peripheral blood monocytes (PBM). Studying dysferlin in monocytes is used for the diagnosis of dysferlin myopathies. The aim of the study was to determine whether dysferlin expression in PBM correlates with that in skeletal muscle.

Methodology/Principal Findings

Using western-blot (WB) we quantified dysferlin expression in PBM from 21 pathological controls with other myopathies in whom mutations in DYSF were excluded and from 17 patients who had dysferlinopathy and two mutations in DYSF. Results were compared with protein expression in muscle by WB and immunohistochemistry (IH). We found a good correlation between skeletal muscle and monocytes using WB. However, IH results were misleading because abnormal expression of dysferlin was also observed in 13/21 pathological controls.

Conclusions/Significance

The analysis of dysferlin protein expression in PBM is helpful when: 1) the skeletal muscle IH pattern is abnormal or 2) when muscle WB can not be performed either because muscle sample is lacking or insufficient or because the muscle biopsy is taken from a muscle at an end-stage and it mainly consists of fat and fibrotic tissue.     

Activity of maize transglutaminase overexpressed in Escherichia coli inclusion bodies: an alternative to protein refolding

Transglutaminases (TGases) catalyze protein post-translational modification by ε-(γ-glutamyl) links and covalent polyamine conjugation. In plants, this enzyme is poorly characterized and only the maize plastidial TGase gene (tgz) has been cloned. The tgz gene (Patent WWO03102128) had been subcloned and overexpressed in Escherichia coli cells, and the recombinant protein (TGZp) was present mainly in inclusion bodies (IB) fraction. In this work, after overexpression of TGZ15p and SDS-PAGE IB fraction analysis, bands about 65 and 56 kDa were obtained. Western blot, alkylation and MALDI-TOF/TOF analyses indicated that the 56 kDa band corresponded to a truncated sequence from the native TGZ15p (expected MW 65 kDa), by elimination of a chloroplast signal peptide fragment during expression processing. So that large-scale protein production and protein crystallization can be applied, we characterized the TGZ15p enzyme activity in the IB protein fraction, with and without refolding. Results indicate that it presented the biochemical characteristics of other described TGases, showing a certain plant-substrate preference. Solubilization of the IB fraction with Triton X-100 as nondenaturing detergent yielded active TGZ without the need for refolding, giving activity values comparable to those of the refolded protein, indicating that this is a valuable, faster way to obtain TGZ active protein.     

Cost-effectiveness of early treatment with first-line NNRTI-based HAART regimens in the UK, 1996-2006

Aim

Calculate time to first-line treatment failure, annual cost and cost-effectiveness of NNRTI versus PIboosted first-line HAART regimens in the UK, 1996–2006.

Background

Population costs for HIV services are increasing in the UK and interventions need to be effective and efficient to reduce or stabilize costs. 2NRTIs + NNRTI regimens are cost-effective regimens for first-line HAART, but these regimens have not been compared with first-line PI_boosted regimens.

Methods

Times to first-line treatment failure and annual costs were calculated for first-line HAART regimens by CD4 count when starting HAART (2006 UK prices). Cost-effectiveness of 2NRTIs+NNRTI versus 2NRTIs+PI_boosted regimens was calculated for four CD4 strata.

Results

55% of 5,541 people living with HIV (PLHIV) started HAART with CD4 count ≤200 cells/mm3, many of whom were Black Africans. Annual treatment cost decreased as CD4 count increased; most marked differences were observed between starting HAART with CD4 ≤200 cells/mm3 compared with CD4 count >200 cells/mm3. 2NRTI+PI_boosted and 2NRTI+NNRTI regimens were the most effective regimens across the four CD4 strata; 2NRTI+NNRTI was cost-saving or cost-effective compared with 2NRTI + PI_boosted regimens.

Conclusion

To ensure more effective and efficient provision of HIV services, 2NRTI+NNRTI should be started as first-line HAART regimen at CD4 counts ≤350 cell/mm3, unless specific contra-indications exist. This will increase the number of PLHIV receiving HAART and will initially increase population costs of providing HIV services. However, starting PLHIV earlier on cost-effective regimens will maintain them in better health and use fewer health or social services, thereby generating fewer treatment and care costs, enabling them to remain socially and economically active members of society. This does raise a number of ethical issues, which will have to be acknowledged and addressed, especially in countries with limited resources.