A Systematic Approach to Mapping Recessive Disease Genes in Individuals from Outbred Populations

The identification of recessive disease-causing genes by homozygosity mapping is often restricted by lack of suitable consanguineous families. To overcome these limitations, we apply homozygosity mapping to single affected individuals from outbred populations. In 72 individuals of 54 kindred ascertained worldwide with known homozygous mutations in 13 different recessive disease genes, we performed total genome homozygosity mapping using 250,000 SNP arrays. Likelihood ratio Z-scores (ZLR) were plotted across the genome to detect ZLR peaks that reflect segments of homozygosity by descent, which may harbor the mutated gene. In 93% of cases, the causative gene was positioned within a consistent ZLR peak of homozygosity. The number of peaks reflected the degree of inbreeding. We demonstrate that disease-causing homozygous mutations can be detected in single cases from outbred populations within a single ZLR peak of homozygosity as short as 2 Mb, containing an average of only 16 candidate genes. As many specialty clinics have access to cohorts of individuals from outbred populations, and as our approach will result in smaller genetic candidate regions, the new strategy of homozygosity mapping in single outbred individuals will strongly accelerate the discovery of novel recessive disease genes.     

Localization and extinction of bacterial populations under inhomogeneous growth conditions

The transition from localized to systemic spreading of bacteria, viruses, and other agents is a fundamental problem that spans medicine, ecology, biology, and agriculture science. We have conducted experiments and simulations in a simple one-dimensional system to determine the spreading of bacterial populations that occurs for an inhomogeneous environment under the influence of external convection. Our system consists of a long channel with growth inhibited by uniform ultraviolet (UV) illumination except in a small "oasis", which is shielded from the UV light. To mimic blood flow or other flow past a localized infection, the oasis is moved with a constant velocity through the UV-illuminated "desert". The experiments are modeled with a convective reaction-diffusion equation. In both the experiment and model, localized or extinct populations are found to develop, depending on conditions, from an initially localized population. The model also yields states where the population grows everywhere. Further, the model reveals that the transitions between localized, extended, and extinct states are continuous and nonhysteretic. However, it does not capture the oscillations of the localized population that are observed in the experiment.     

Sulfate activation in Desulfotomaculum

Enzyme activities conceivably involved in the activation of sulfate were studied with Desulfotomaculum ruminis, D. acetoxidans, D. nigrificans, D. orientis, and Desulfovibrio vulgaris. Cell lysates of these species revealed activities of at least 8 nkat/mg protein (i.e., 480 nmol per min and mg protein) of ATP sulfurylase, acetate kinase, phosphotransacetylase and adenylate kinase. ADP sulfurylase was not detected. Pyrophosphatase activity was high (73 to 97 nkat/mg protein) in Desulfotomaculum orientis and Desulfovibrio vulgaris. In these strains pyrophosphatase was activated by addition of a reductant (dithionite). In Desulfotomaculum ruminis, D. acetoxidans, and D. nigrificans, only low pyrophosphatase activity (2.5 to 6.3 nkat/mg protein) was measured, which was not reductant-activated. Some hints indicated a membrane association of the pyrophosphatase in D. ruminis, and possibly also in D. acetoxidans and D. nigrificans. Activities of a pyrophosphate-dependent acetate kinase (PP_i:acetate kinase), a PP_i:AMP kinase or a polyphosphate:AMP kinase were not detected or negligible. The results are not in favour of the assumption that pyrophosphate formed by ATP sulfurylase during sulfate activation might be utilized to form acetyl phosphate in Desulfotomaculum species. Contrary results of other authors were shown to be artefacts caused by chemical hydrolysis of acetyl phosphate in the molybdate-sulfuric acid reagent used for phosphate determination.Abbreviations P_i orthophosphate - PP_i pyrophosphate - APS adenosine phosphosulfate - AP₅A, P¹ P⁵-di(adenosine-5-)pentaphosphate - CTAB cetyltrimethylammonium bromide - MOPS 3-(N-morpholino)propanesulfonic acid - HEPES N(-2-hydroxyethyl)piperazine-N-2-ethanesulfonic acid     

Genetic improvement of Bacillus licheniformis strains for efficient deproteinization of shrimp shells and production of high-molecular-mass chitin and chitosan

By targeted deletion of the polyglutamate operon (pga) in Bacillus licheniformis F11, a derivative form, F11.1 (Δpga), was obtained that, along with lacking polyglutamate (PGA) formation, displayed enhanced proteolytic activities. The phenotypic properties were maintained in a strain in which the chiBA operon was additionally deleted: F11.4 (ΔchiBA Δpga). These genetically modified strains, carrying the Δpga deletion either alone (F11.1) or together with the ΔchiBA (F11.4) deletion, were used in fermentations (20-liter scale) aiming at the deproteinization of shrimp shells in order to obtain long-chain chitin. After chemical deacetylation, the resulting chitosan samples were analyzed by nuclear magnetic resonance spectroscopy, size exclusion chromatography, and viscometry and compared to a chitosan preparation that was produced in parallel by chemical methods by a commercial chitosan supplier (GSRmbH). Though faint lipid impurities were present in the fermented polysaccharides, the viscosity of the material produced with the double-deletion mutant F11.4 (Δpga ΔchiBA) was higher than that of the chemically produced and commercially available samples (Cognis GmbH). Thus, enhanced proteolytic activities and a lack of chitinase activity render the double mutant F11.4 a powerful tool for the production of long-chain chitosan.     

Dialogue and collaboration with ECVAM: the view of the EFPIA

An evaluation is presented of past experience of dialogue and collaboration of ECVAM with the European Federation of Pharmaceutical Industries and Associations (EFPIA) over the last nine years. Lessons learnt from the viewpoint of EFPIA company representatives are given. Also, proposals for the future ECVAM approach are made, such as support for other research areas for new methods to be validated, giving realistic statements to ECVAM's EU and external customers, and being open to any new technology development that might help in opening and establishing new alternative avenues. Finally, the need for proper publications on the implementation of alternatives is recommended, for example, through the existing national platforms and their umbrella organisation, ecopa.     

Physiology of polyol formation by free and immobilized cells of the osmotolerant yeast Pichia farinosa

Summary Some physiological data of cells of Pichia farinosa immobilized on sintered glass Raschig rings were compared with data from free cells. Glucose consumption and productivity of total polyols (arabitol, glycerol and erythritol) showed a simultaneous inter-lag phase. Enzymes that catalyse steps of the pentosephosphate pathway (glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, transaldolase and polyol dehydrogenase) showed a distinct increase after transfer of the cells into production medium. The activity of glycerol-3-phosphate dehydrogenase was generally low. Only alcohol dehydrogenase presented the inter-lag phase mentioned above.Offprint requests to: H.-J. Rehm     

Interaction between shock coils increased the incidence of inappropriate therapies and lead failure in implantable cardioverter defibrillator

Aims

Shock coil interaction in patients with multiple implantable cardioverter defibrillator (ICD) leads is occasionally observed. We aimed to evaluate the incidence of shock coil interaction and its clinical relevance.

A microsphere study on the effects of somatostatin and secretin on regional blood flow in anesthetized dogs

The endogenous peptides somatostatin and secretin are effective in the therapy of upper gastrointestinal tract bleeding and acute pancreatitis. The clinical effects may be partly brought about by changes in the regional blood flow. To evaluate the effects of somatostatin (50 and 100 μg/min over 6–8 min) and secretin (0.1 and 0.5 U · kg^?1 · min^?1 over 3–5 min) on tissue blood flow, particularly of the gastrointestinal tract, the tracer microsphere reference sample method was used in anesthetized dogs.Infusion of somatostatin significantly diminished gastric and pancreatic blood flow whereas no changes of duodenal and ileal blood flow could be obtained. Blood flow through spleen, kidneys and adrenal glands was increased but no changes were observed in the blood flow of other tissues. Cardiac hemodynamics remained unchanged.Secretin increased the blood flow of the duodenum, the kidneys and the adrenal glands and diminished gastric blood flow without changing pancreatic, ileal, hepatic, pulmonary and muscle blood flow. Cerebral, pituitary and myocardial blood flow was increased by a higher dose of secretin. It also evoked a slight but significant positive ino- and chronotropic effect. Since secretin and somatostatin differ in their respective effects on gastrointestinal blood flow it is suggested that the previously reported beneficial effects of both peptides on upper gastrointestinal bleeding cannot solely be attributed to changes in regional blood flow.     

Angiotensin converting enzyme inhibitors,left ventricular hypertrophy and fibrosis

From pharmacological investigations and clinical studies, it is known that angiotensin converting enzyme (ACE) inhibitors exhibit additional local actions, which are not related to hemodynamic changes and which cannot be explained only by interference with the renin angiotensin system (RAS) by means of an inhibition of angiotensin II (ANG II) formation. Since ACE is identical to kininase II, which inactivates the nonapeptide bradykinin (BK) and related kinins, potentiation of kinins might be responsible for these additional effects of ACE inhibitors.

1. In rats made hypertensive by aortic banding, the effect of ramipril in left ventricular hypertrophy (LVH) was investigated. Ramipril in the antihypertensive dose of 1 mg/kg/day for 6 weeks prevented the increase in blood pressure and the development of LVH. The low dose of ramipril (10 μg/kg/day for 6 weeks) had no effect on the increase in blood pressure or on plasma ACE activity but also prevented LVH after aortic banding. The antihypertrophic effect of the higher and lower doses of ramipril, as well as the antihypertensive action of the higher dose of ramipril, was abolished by coadmistration of the kinin receptor antagonist icatibant. In the regression study the antihypertrophic actions of ramipril were not blocked by the kinin receptor antagonist. Chronic administration of BK had similar beneficial effects in a prevention study which were abolished by icatibant and N^G-nitro-L-arginine (L-NNA). In a one year study the high and low dose of ramipril prevented LVH and fibrosis. Ramipril had an early direct effect in hypertensive rats on the mRNA expression for myocardial collagen I and III, unrelated to its blood pressure lowering effect.
2. In spontaneously hypertensive rats (SHR) the preventive effects of chronic treatment with ramipril on myocardial LVH was investigated. SHR were treated in utero and, subsequently, up to 20 weeks of age with a high dose (1 mg/kg/day) or with a low dose (10 μg/kg/day) of ramipril. Animals on a high dose remained normotensive, whereas those on a low dose developed hypertension in parallel to vehicle-treated controls. Left ventricular mass was reduced only in high-dose-treated, but not in low-dose treated animals but both groups revealed an increase in myocardial capillary length density. In SHR stroke prone animals cardiac function and metabolism was improved by ramipril and abolished by coadministration of icatibant. In contrast to the prevention studies, in a regression study ramipril reduced cardiac hypertrophy also by low dose treatment.
3. In rats chronic nitric oxide (NO) inhibition by N^G-nitro-L-arginine-methyl ester (L-NAME) treatment induced hypertension and LVH. Ramipril protected against blood pressure increase and partially against myocardial hypertrophy.

These experimental findings in different models of LVH characterise ACE inhibitors as remarkable antihypertrophic and antifibrotic substances.