The molecular basis of cystathionine beta-synthase deficiency in Dutch patients with homocystinuria: effect of CBS genotype on biochemical and clinical phenotype and on response to treatment.

Homocystinuria due to cystathionine beta-synthase (CBS) deficiency, inherited as an autosomal recessive trait, is the most prevalent inborn error of methionine metabolism. Its diverse clinical expression may include ectopia lentis, skeletal abnormalities, mental retardation, and premature arteriosclerosis and thrombosis. This variability is likely caused by considerable genetic heterogeneity. We investigated the molecular basis of CBS deficiency in 29 Dutch patients from 21 unrelated pedigrees and studied the possibility of a genotype-phenotype relationship with regard to biochemical and clinical expression and response to homocysteine-lowering treatment. Clinical symptoms and biochemical parameters were recorded at diagnosis and during long-term follow-up. Of 10 different mutations detected in the CBS gene, 833T-->C (I278T) was predominant, present in 23 (55%) of 42 independent alleles. At diagnosis, homozygotes for this mutation (n=12) tended to have higher homocysteine levels than those seen in patients with other genotypes (n=17), but similar clinical manifestations. During follow-up, I278T homozygotes responded more efficiently to homocysteine-lowering treatment. After 378 patient-years of treatment, only 2 vascular events were recorded; without treatment, at least 30 would have been expected (P<.01). This intervention in Dutch patients significantly reduces the risk of cardiovascular disease and other sequelae of classical homocystinuria syndrome.     

Cyclic Expression of A Nuclear Protein In A Dinoflagellate

Nuclei of the dinoflagellate Crypthecodinium cohnii strain Whd were isolated and nuclear proteins were extracted in three fractions, corresponding to the increasing affinity of these proteins to genomic DNA. One fraction contained two major bands (48- and 46-kDa) and antibodies specific to this fraction revealed two major bands by Western blot on nuclear extracts, corresponding to the 46- and 48-kDa bands. The 48-kDa protein was detected in G1 phase but not in M phase cells. An expression cDNA library of C. cohnii was screened with these antibodies, and two different open reading frames were isolated. Dinoflagellate nuclear associated protein (Dinap1), one of these coding sequences, was produced in E. coli and appeared to correspond to the 48-kDa nuclear protein. No homologue of this sequence was found in the data bases, but two regions were identified, one including two putative zinc finger repeats, and one coding for two potential W/W domains. The second coding sequence showed a low similarity to non-specific sterol carrier proteins. Immunocytolocalization with specific polyclonal antibodies to recombinant Dinap1 showed that the nucleus was immunoreactive only during the G1 phase: the nucleoplasm was immunostained, while chromosome cores and nuclear envelopes were negative.     

Large-scale identification of polymorphic microsatellites using an <Emphasis Type="Italic">in silico</Emphasis> approach

Background  

Simple Sequence Repeat (SSR) or microsatellite markers are valuable for genetic research. Experimental methods to develop SSR markers are laborious, time consuming and expensive. In silico approaches have become a practicable and relatively inexpensive alternative during the last decade, although testing putative SSR markers still is time consuming and expensive. In many species only a relatively small percentage of SSR markers turn out to be polymorphic. This is particularly true for markers derived from expressed sequence tags (ESTs). In EST databases a large redundancy of sequences is present, which may contain information on length-polymorphisms in the SSR they contain, and whether they have been derived from heterozygotes or from different genotypes. Up to now, although a number of programs have been developed to identify SSRs in EST sequences, no software can detect putatively polymorphic SSRs.     

Carbon accumulation in agricultural soils after afforestation: a meta-analysis

Deforestation usually results in significant losses of soil organic carbon (SOC). The rate and factors determining the recovery of this C pool with afforestation are still poorly understood. This paper provides a review of the influence of afforestation on SOC stocks based on a meta-analysis of 33 recent publications (totaling 120 sites and 189 observations), with the aim of determining the factors responsible for the restoration of SOC following afforestation. Based on a mixed linear model, the meta-analysis indicates that the main factors that contribute to restoring SOC stocks after afforestation are: previous land use, tree species planted, soil clay content, preplanting disturbance and, to a lesser extent, climatic zone. Specifically, this meta-analysis (1) indicates that the positive impact of afforestation on SOC stocks is more pronounced in cropland soils than in pastures or natural grasslands; (2) suggests that broadleaf tree species have a greater capacity to accumulate SOC than coniferous species; (3) underscores that afforestation using pine species does not result in a net loss of the whole soil-profile carbon stocks compared with initial values (agricultural soil) when the surface organic layer is included in the accounting; (4) demonstrates that clay-rich soils (> 33%) have a greater capacity to accumulate SOC than soils with a lower clay content (< 33%); (5) indicates that minimizing preplanting disturbances may increase the rate at which SOC stocks are replenished; and (6) suggests that afforestation carried out in the boreal climate zone results in small SOC losses compared with other climate zones, probably because trees grow more slowly under these conditions, although this does not rule out gains over time after the conversion. This study also highlights the importance of the methodological approach used when developing the sampling design, especially the inclusion of the organic layer in the accounting.     

Comparing the Cervista HPV HR Test and Hybrid Capture 2 Assay in a Dutch Screening Population: Improved Specificity of the Cervista HPV HR Test by Changing the Cut-Off

The diagnostic performance of the widely-used Cervista HPV HR test was compared to the Hybrid Capture 2 (HC2) test in a Dutch population-based cervical cancer screening program. In 900 scrapings of women with normal cytomorphology, specificity was 90% (95%CI: 87.84–91.87) for the Cervista HPV HR test and 96% (95%CI: 94.76–97.37) for the HC2 test with 93% agreement between both tests (κ = 0.5, p<0.001). The sensitivity for CIN2+ using 65 scrapings of women with histological-confirmed CIN2+ was 91% (95%CI: 80.97–96.51) for the Cervista HPV HR test and 92% (95%CI: 82.94–97.43) for the HC2 test with 95% agreement between both tests (κ = 0.7, p<0.001). Fifty-seven of 60 HC2 negative/Cervista positive cases tested HPV-negative with PCR-based HPV assays; of these cases 56% were defined as Cervista triple-positive with FOZ values in all 3 mixes higher than the second cut-off of 1.93 (as set by manufacturer). By setting this cut-off at 5.0, specificity improved significantly without affecting sensitivity. External validation of this new cut-off at 5.0 in triple-positive scrapings of women selected from the SHENCCASTII database revealed that 22/24 histological normal cases now tested HPV-negative in the Cervista HPV HR test, while CIN2+ lesions remained HPV-positive. The intra-laboratory reproducibility of the Cervista HPV HR test (n = 510) showed a concordance of 92% and 93% for cut-off 1.93 and 5.0 (κ = 0.83 and κ = 0.84, p<0.001) and inter-laboratory agreement of the Cervista HPV HR test was 90% and 93% for cut-off 1.93 and 5.0 (κ = 0.80 and κ = 0.85, p<0.001). In conclusion, the specificity of the Cervista HPV HR test could be improved significantly by increasing the second cut-off from 1.93 to 5.0, without affecting the sensitivity of the test in a population-based screening setting.     

Dissociation of intracellular lysosomal rupture from the cell death caused by silica

The relationship between intracellular lysosomal rupture and cell death caused by silica was studied in P388d(1) macrophages. After 3 h of exposure to 150 μg silica in medium containing 1.8 mM Ca(2+), 60 percent of the cells were unable to exclude trypan blue. In the absence of extracellular Ca(2+), however, all of the cells remained viable. Phagocytosis of silica particles occurred to the same extent in the presence or absence of Ca(2+). The percentage of P388D(1) cells killed by silica depended on the dose and the concentration of Ca(2+) in the medium. Intracellular lyosomal rupture after exposure to silica was measured by acridine orange fluorescence or histochemical assay of horseradish peroxidase. With either assay, 60 percent of the cells exposed to 150 μg silica for 3 h in the presence of Ca(2+) showed intracellular lysosomal rupture, was not associated with measureable degradation of total DNA, RNA, protein, or phospholipids or accelerated turnover of exogenous horseradish peroxidase. Pretreatment with promethazine (20 μg/ml) protected 80 percent of P388D(1) macrophages against silica toxicity although lysosomal rupture occurred in 60-70 percent of the cells. Intracellular lysosomal rupture was prevented in 80 percent of the cells by pretreatment with indomethacin (5 x 10(-5)M), yet 40-50 percent of the cells died after 3 h of exposure to 150 μg silica in 1.8 mM extracellular Ca(2+). The calcium ionophore A23187 also caused intracellular lysosomal rupture in 90-98 percent of the cells treated for 1 h in either the presence or absence of extracellular Ca(2+). With the addition of 1.8 mM Ca(2+), 80 percent of the cells was killed after 3 h, whereas all of the cells remained viable in the absence of Ca(2+). These experiments suggest that intracellular lysosomal rupture is not causally related to the cell death cause by silica or {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Cell death is dependent on extracellular Ca(2+) and may be mediated by an influx of these ions across the plasma membrane permeability barrier damaged directly by exposure to these toxins.     

Bootstrapping phylogenies inferred from rearrangement data

ABSTRACT: Large-scale sequencing of genomes has enabled the inference of phylogenies based on the evolution of genomic architecture, under such events as rearrangements, duplications, and losses. Many evolutionary models and associated algorithms have been designed over the last few years and have found use in comparative genomics and phylogenetic inference. However, the assessment of phylogenies built from such data has not been properly addressed to date. The standard method used in sequence-based phylogenetic inference is the bootstrap, but it relies on a large number of homologous characters that can be resampled; yet in the case of rearrangements, the entire genome is a single character. Alternatives such as the jackknife suffer from the same problem, while likelihood tests cannot be applied in the absence of well established probabilistic models. We present a new approach to the assessment of distance-based phylogenetic inference from whole-genome data; our approach combines features of the jackknife and the bootstrap and remains nonparametric. For each feature of our method, we give an equivalent feature in the sequence-based framework; we also present the results of extensive experimental testing, in both sequence-based and genome-based frameworks. Through the feature-by-feature comparison and the experimental results, we show that our bootstrapping approach is on par with the classic phylogenetic bootstrap used in sequence-based reconstruction, and we establish the clear superiority of the classic bootstrap for sequence data and of our corresponding new approach for rearrangement data over proposed variants. Finally, we test our approach on a small dataset of mammalian genomes, verifying that the support values match current thinking about the respective branches. Our method is the first to provide a standard of assessment to match that of the classic phylogenetic bootstrap for aligned sequences. Its support values follow a similar scale and its receiver-operating characteristics are nearly identical, indicating that it provides similar levels of sensitivity and specificity. Thus our assessment method makes it possible to conduct phylogenetic analyses on whole genomes with the same degree of confidence as for analyses on aligned sequences. Extensions to search-based inference methods such as maximum parsimony and maximum likelihood are possible, but remain to be thoroughly tested.     

The natural history of homocystinuria due to cystathionine beta-synthase deficiency

An international questionnaire survey has been conducted to define better the natural history of homocystinuria due to cystathionine beta-synthase deficiency and permit evaluation of treatment. Data were compiled for 629 patients. Among patients not discovered by newborn screening, B6-responsive individuals on the average have significantly better mental capabilities (mean IQ, 79) than do B6-nonresponsive individuals (mean IQ, 57). Time-to-event curves are presented for the other major clinical abnormalities produced by this disease. Each occurred at significantly lower rates in untreated B6-responsive than in untreated B6-nonresponsive patients, as shown by the following examples: (1) dislocation of optic lenses (at age 10, chances of dislocation: 55% and 82%, respectively); (2) initial clinically detected thromboembolic events (at age 15, chances of having had such an event: 12% and 27%, respectively); (3) radiologic detection of spinal osteoporosis (at age 15, chances of such osteoporosis having been detected: 36% and 64%, respectively); and (4) mortality (at age 30, chances of not surviving: 4% and 23%, respectively). Methionine restriction initiated neonatally prevented mental retardation, retarded the rate of lens dislocation, and may have reduced the incidence of seizures. Pyridoxine treatment of late-detected B6-responsive patients retarded the rate of occurrence of initial thromboembolic events. Following 586 surgical procedures, 25 postoperative thromboembolic complications occurred, six of which were fatal. Reproductive histories were reported predominantly for B6-responsive patients. Living offspring of either men or women patients had few abnormalities. The evidence is inconclusive whether untreated maternal cystathionine beta-synthase deficiency leads to excessive fetal loss. Only 13% of patients detected in screening programs of newborns and classified as to B6-responsiveness were B6-responsive, compared to 47% among late-detected patients. Current screening programs that identify neonatal hypermethioninemia may be preferentially failing to detect B6-responsive patients.