Nichtinvasive molekulargenetische Methoden in der pränatalen Diagnostik

Work on the development of noninvasive prenatal tests to avoid risk to the fetus in traditional amniocentesis or chorion villus biopsy has been ongoing for many years. Until recently, most approaches were extremely expensive and limited only to selected applications, thus they failed to develop beyond a “proof-of-principle” status. This has changed radically as a result of the introduction of new sequencing methods, since initial studies have shown that fetal aneuploidies from maternal plasma DNA can be identified correctly. In addition, these techniques make it possible to establish even the mutation status of the fetus. While on the one hand this offers completely new options in prenatal diagnosis, progress of this kind is associated with significant ethical challenges on the other. This overview article presents the development of these new methods.     

ADAPTATION OF CUTANEOUS TACTILE RECEPTORS. II

Studies of axon potentials set up by pressure stimuli applied to single cutaneous receptors in frog''s skin indicate that the mechanical stimulus excites the free nerve endings directly. Adaptation to constant pressures or to intermittently applied pressures (failure of the response) may be due to the reduction of excitability of the nerve endings by potassium released under the pressure from surrounding epithelial cells.     

ACCUMULATION OF SALT AND PERMEABILITY IN PLANT CELLS

1. Comparisons are made of concentrations of K and Br in exudates of barley roots and in expressed sap from roots, under conditions favorable for aerobic metabolism. Both methods lead to the same general viewpoint concerning metabolically governed transport of solutes by living plant cells. 2. Cyanide in low concentration prevented salt accumulation by barley roots. Methylene blue, without decrease of CO₂ production by roots, destroyed power of salt accumulation. 3. K and Br ions entered roots to only a slight extent under an anaerobic condition, even with an inward gradient of ionic concentration. 4. Lactate or alcohol, under aerobic conditions, did not prevent rapid accumulation of salt by root cells. 5. Experiments on fluids obtained by suction from tomato roots gave evidence of loss of salt-accumulating power under the influence of N₂ gas or CO₂ gas, together with probable effects on cell permeability. 6. Several experiments on Nitella cells in which radioactive isotopes were used are reported. Bromide gradually moved into vacuolar sap until the concentration appeared to exceed that of the protoplasm, on the basis of the results of the several types of experiments. Accumulation of salt in the vacuole did not occur anaerobically. 7. Some views of interrelations of permeability, salt accumulation, and metabolism are suggested for further discussion.     

QUANTITATIVE ASPECTS OF CUTANEOUS SENSORY ADAPTATION. I

1. Adaptation of tactile receptors in the skin of the frog to excitation by an intermittent jet of air is measured and correlated with certain properties of a series of notched discs used to interrupt the air stream. 2. Adaptation in fifteen cases is found to be described by either one of two empirical formulas, or t = -k log f + C, for nine preparations t = a f^-b, for six preparations where f is the per cent frequency at time t and -k and -b are constants defining the rate of adaptation.     

Die Genetik der bipolaren Störung

With a life-time prevalence of about 0.5–1.5%, bipolar (manic depressive) disorder represents a common psychiatric disease. Family, twin, and adoption studies have consistently shown that genetic factors contribute to disease development. Genome-wide linkage studies have detected chromosomal regions that are very likely to harbor predisposing genes. Meta-analyses suggest, however, that the genetic contribution of the individual loci must be relatively small which could be one reason for the difficulties in identifying the genes responsible. Very recently, genome-wide association analyses, investigating hundreds of thousands of single nucleotide polymorphisms (SNPs) in phenotypically well-characterized patient and control cohorts, promise a major breakthrough in search of disease-associated genes.     

Genetik und Klinik der Hämophilie A und B

Haemophilia A and B are caused by various mutations in the factor VIII (FVIII) and factor IX (FIX) genes, respectively. The clinical course of the disease is variable, dependent on the severity of the molecular defect. Nowadays, haemophilia patients can excellently be treated by plasma-derived or recombinant clotting factor concentrates. Thus, bleeding and its consequences can be almost completely prevented with nearly normal quality of life and life expectancy. The most severe complication of this treatment is the formation of antibodies (inhibitors) against the substituted clotting factor. The risk of inhibitor formation correlates significantly with specific mutation types that preclude endogenous factor VIII/IX protein synthesis and can be as high as 20–50%. The information on the expected clinical course is at present the most important indication for FVIII/IX gene analysis. Knowledge of the underlying FVIII/IX gene mutation further allows a reliable and fast carrier diagnosis in female relatives of patients with haemophilia.     

Expression in Escherichia coli, purification, refolding and antifungal activity of an osmotin from Solanum nigrum

Background

The large-scale production of G-protein coupled receptors (GPCRs) for functional and structural studies remains a challenge. Recent successes have been made in the expression of a range of GPCRs using Pichia pastoris as an expression host. P. pastoris has a number of advantages over other expression systems including ability to post-translationally modify expressed proteins, relative low cost for production and ability to grow to very high cell densities. Several previous studies have described the expression of GPCRs in P. pastoris using shaker flasks, which allow culturing of small volumes (500 ml) with moderate cell densities (OD600 ~15). The use of bioreactors, which allow straightforward culturing of large volumes, together with optimal control of growth parameters including pH and dissolved oxygen to maximise cell densities and expression of the target receptors, are an attractive alternative. The aim of this study was to compare the levels of expression of the human Adenosine 2A receptor (A_2AR) in P. pastoris under control of a methanol-inducible promoter in both flask and bioreactor cultures.

Results

Bioreactor cultures yielded an approximately five times increase in cell density (OD600 ~75) compared to flask cultures prior to induction and a doubling in functional expression level per mg of membrane protein, representing a significant optimisation. Furthermore, analysis of a C-terminally truncated A_2AR, terminating at residue V334 yielded the highest levels (200 pmol/mg) so far reported for expression of this receptor in P. pastoris. This truncated form of the receptor was also revealed to be resistant to C-terminal degradation in contrast to the WT A_2AR, and therefore more suitable for further functional and structural studies.

Conclusion

Large-scale expression of the A_2AR in P. pastoris bioreactor cultures results in significant increases in functional expression compared to traditional flask cultures.     

Iron-mediated inhibition of mitochondrial manganese uptake mediates mitochondrial dysfunction in a mouse model of hemochromatosis

Previous phenotyping of glucose homeostasis and insulin secretion in a mouse model of hereditary hemochromatosis (Hfe(-/-)) and iron overload suggested mitochondrial dysfunction. Mitochondria from Hfe(-/-) mouse liver exhibited decreased respiratory capacity and increased lipid peroxidation. Although the cytosol contained excess iron, Hfe(-/-) mitochondria contained normal iron but decreased copper, manganese, and zinc, associated with reduced activities of copper-dependent cytochrome c oxidase and manganese-dependent superoxide dismutase (MnSOD). The attenuation in MnSOD activity was due to substantial levels of unmetallated apoprotein. The oxidative damage in Hfe(-/-) mitochondria is due to diminished MnSOD activity, as manganese supplementation of Hfe(-/-) mice led to enhancement of MnSOD activity and suppressed lipid peroxidation. Manganese supplementation also resulted in improved insulin secretion and glucose tolerance associated with increased MnSOD activity and decreased lipid peroxidation in islets. These data suggest a novel mechanism of iron-induced cellular dysfunction, namely altered mitochondrial uptake of other metal ions.     

Non-traditional tools for lca and sustainability

LCA practice focuses on impacts resulting from the release of chemicals into the environment, but consideration of ‘non-chemical impacts’ is as important for LCA, particularly as it relates to sustainability. Methodologies and philosophies exist for addressing non-chemical impacts, particularly in the area of resource depletion and land use, but the problem of comparing or integrating chemical and non-chemical impacts remains. A new approach for identifying and integrating impacts involves the use of an object-oriented modeling and simulation platform, such as Department of Energy Argonne National Laboratory’s Dynamic Information Architecture System (DIAS). LCA and impact categories can be described as ‘objects’ (at any level of detail or specificity) and any combination of objects and behaviors can be brought into a DIAS analysis frame. Related models that address objects’ behavior characteristics are linked only to their respective objects, not to each other. Thus, maximum flexibility and speed is possible. The process of dividing LCA and impact assessment into a hierarchy of objects provides new insights into the complex mixture of dynamic things, activities, and relationships inherent in LCA and sustainability. Ultimately, embracing the complexity of LCA may be the way to simplify it.