Interaction between the Msh2 and Msh6 Nucleotide-binding Sites in the Saccharomyces cerevisiae Msh2-Msh6 Complex

Indirect evidence has suggested that the Msh2-Msh6 mispair-binding complex undergoes conformational changes upon binding of ATP and mispairs, resulting in the formation of Msh2-Msh6 sliding clamps and licensing the formation of Msh2-Msh6-Mlh1-Pms1 ternary complexes. Here, we have studied eight mutant Msh2-Msh6 complexes with defective responses to nucleotide binding and/or mispair binding and used them to study the conformational changes required for sliding clamp formation and ternary complex assembly. ATP binding to the Msh6 nucleotide-binding site results in a conformational change that allows binding of ATP to the Msh2 nucleotide-binding site, although ATP binding to the two nucleotide-binding sites appears to be uncoupled in some mutant complexes. The formation of Msh2-Msh6-Mlh1-Pms1 ternary complexes requires ATP binding to only the Msh6 nucleotide-binding site, whereas the formation of Msh2-Msh6 sliding clamps requires ATP binding to both the Msh2 and Msh6 nucleotide-binding sites. In addition, the properties of the different mutant complexes suggest that distinct conformational states mediated by communication between the Msh2 and Msh6 nucleotide-binding sites are required for the formation of ternary complexes and sliding clamps.     

Independence between modification of genetic position effects and formation of lampbrush loops by the Y chromosome of Drosophila hydei

Summary The phenotype of the variegation position effect white-mottled-2 in Drosophila hydei is modified by supernumerary Y chromosomes and by fractions thereof. Different translocated Y fragments have varying degrees of effectiveness in suppressing the mutant phenotype in the mottled eyes. In fragments derived from similar regions of the Y chromosome the suppressive ability is related to their cytological lengths. In contrast, fragments derived from distinctive regions of the Y chromosome differ markedly in their effectiveness, and these differences are not necessarily correlated with the cytological length. In particular, fragments of the distal region of Y^L are more effective in enhancing the wild phenotype than are proximal fragments of similar size.The mutation white-mottled-2 is accompanied by a complex rearrangement of the X chromosome. This inhibits crossing over between large regions of the X chromosome in structural heterozygotes; it causes also a delay of development and a considerable reduction of viability in homozygous females and hemizygous males. XO males are inviable. The inviability of these males is partially covered by Y fragments. With respect to viability, the fragments show similar regional differences in effectiveness as in the modification of the mottled phenotype.There is also a parental effect on the modulation of the white-mottled-2 phenotype.There is no correlation between the activity of Y chromosomal factors on spermiogenesis and the activity of Y factors on the modification of the variegation position effect. Suppression of Y chromosomal sites which normally unfold lampbrush loops during the spermatocyte stage and whose activity has previously been shown to be indispensible for normal differentiation of the male germ line cells does not result in any visible alterations of the effectiveness on the mottling. So there is obviously independence between these two different genetic activities of Y chromosomal factors.     

‘Mild mitochondrial uncoupling’ induced protection against neuronal excitotoxicity requires AMPK activity

The preconditioning response conferred by a mild uncoupling of the mitochondrial membrane potential (Δψ_m) has been attributed to altered reactive oxygen species (ROS) production and mitochondrial Ca^2 + uptake within the cells. Here we have explored if altered cellular energetics in response to a mild mitochondrial uncoupling stimulus may also contribute to the protection. The addition of 100 nM FCCP for 30 min to cerebellar granule neurons (CGNs) induced a transient depolarization of the Δψ_m, that was sufficient to significantly reduce CGN vulnerability to the excitotoxic stimulus, glutamate. On investigation, the mild mitochondrial ‘uncoupling’ stimulus resulted in a significant increase in the plasma membrane levels of the glucose transporter isoform 3, with a hyperpolarisation of Δψ_m and increased cellular ATP levels also evident following the washout of FCCP. Furthermore, the phosphorylation state of AMP-activated protein kinase (AMPK) (Thr 172) was increased within 5 min of the uncoupling stimulus and elevated up to 1 h after washout. Significantly, the physiological changes and protection evident after the mild uncoupling stimulus were lost in CGNs when AMPK activity was inhibited. This study identifies an additional mechanism through which protection is mediated upon mild mitochondrial uncoupling: it implicates increased AMPK signalling and an adaptive shift in energy metabolism as mediators of the preconditioning response associated with FCCP-induced mild mitochondrial uncoupling.     

Maximization of pulmonary hygroscopic aerosol deposition

A newly developed computer model is used to predict the aqueous salt solution concentration, breathing pattern, and inhaled droplet size distribution parameters that will maximize pulmonary deposition of hygroscopic medicinal aerosols. The parameter values providing maximum pulmonary deposition include 1) a NaCl concentration in the aerosolized solution of 0.035 g/ml or higher if the subject can tolerate it, 2) as nearly a monodispersed inhaled aerosol size distribution as possible, 3) an aerosol mass median diameter of 2-3 micron, and 4) slow (7 breaths/min) uninterrupted breathing of 1.5-2 liters of aerosol/breath. With these values, the model predicts that pulmonary deposition can be increased by greater than 100% relative to the deposition achieved in conventional inhalation therapy with isotonic saline-based medications.     

State-of-the art data normalization methods improve NMR-based metabolomic analysis

Extracting biomedical information from large metabolomic datasets by multivariate data analysis is of considerable complexity. Common challenges include among others screening for differentially produced metabolites, estimation of fold changes, and sample classification. Prior to these analysis steps, it is important to minimize contributions from unwanted biases and experimental variance. This is the goal of data preprocessing. In this work, different data normalization methods were compared systematically employing two different datasets generated by means of nuclear magnetic resonance (NMR) spectroscopy. To this end, two different types of normalization methods were used, one aiming to remove unwanted sample-to-sample variation while the other adjusts the variance of the different metabolites by variable scaling and variance stabilization methods. The impact of all methods tested on sample classification was evaluated on urinary NMR fingerprints obtained from healthy volunteers and patients suffering from autosomal polycystic kidney disease (ADPKD). Performance in terms of screening for differentially produced metabolites was investigated on a dataset following a Latin-square design, where varied amounts of 8 different metabolites were spiked into a human urine matrix while keeping the total spike-in amount constant. In addition, specific tests were conducted to systematically investigate the influence of the different preprocessing methods on the structure of the analyzed data. In conclusion, preprocessing methods originally developed for DNA microarray analysis, in particular, Quantile and Cubic-Spline Normalization, performed best in reducing bias, accurately detecting fold changes, and classifying samples.

     

Electrooptical analysis of blue and of cation-regenerated bacteriorhodopsin

Blue bacteriorhodopsin was prepared by electrodialysis, cation-exchange chromatography and acidification. The electrooptical properties of these preparations compared to those of the native purple bacteriorhodopsin suggest that the blue bacteriorhodopsin has a smaller induced dipole moment than the native purple bacteriorhodopsin and that bound cations in the native bacteriorhodopsin stabilize the protein conformation in the membrane.Purple bacteriorhodopsin was regenerated by addition of potassium, magnesium or ferric ions to blue bacteriorhodopsin. Both spectrscopically and electrooptically the potassium- and ferric-regenerated samples are different from the native purple state. Although the magnesium-regenerated sample is spectroscopically similar to the native purple bacteriorhodopsin, the electrooptical properties are rather similar to those of the cation-depleted blue sample, suggesting that it is very difficult to re-stabilize protein structures once cations are depleted.     

The fungicide methyl 2-benzimidazole carbamate causes infertility in male Sprague-Dawley rats

A serial breeding technique was used to evaluate the fertility of male Sprague-Dawley rats after exposure to the fungicide carbendazim (methyl 2-benzimidazole carbamate). Proven-fertile male rats (90 days old) received 10 daily doses of corn oil or carbendazim (400 mg/kg/day) peroral. Each male was bred with a new female each week; breeding began on the third day of treatment and continued for 32 wk after the last day of chemical exposure. Twelve days after each breeding period, the females were killed, their uteri were examined for resorptions, and the number of dead and viable fetuses was determined. All males were killed 35 wk post exposure, and testicular tissue was prepared for histopathological examination by vascular perfusion. Fertility (percent fertile as indicated by pregnant females) of males in the carbendazim-treated group was depressed the first post-exposure week; 10 of the 24 treated males failed to produce a pregnant female as compared with no failures in the control group. By the fifth post-exposure week, 16 of the 24 carbendazim-treated males were infertile. Of these 16 males, 4 recovered fertility after a failure to produce a pregnant female for 5-11 consecutive breeding periods. However, 12 of the males did not recover fertility during the remainder of the 32-wk post-exposure period. Histological examinations of testicular sections 245 days post exposure revealed that exposure to carbendazim caused severe seminiferous tubular atrophy (greater than 85% of tubules were atrophic) in those carbendazim-treated males that failed to recover fertility.(ABSTRACT TRUNCATED AT 250 WORDS)     

Three-dimensional representation of chemical gradients

Perspective display techniques are applied to chemical and biochemical data sets. These represent spatially distributed gradients of reactive compounds that participate in pattern-formation processes due to reaction-diffusion or reaction-convection coupling. The patterns form in thin solution layers and are observed as chemical waves in the Belousov-Zhabotinskii reaction, as convection-induced stationary structures during oscillating glycolysis in yeast cytoplasm, and as the diffusive spreading of enzyme-catalyzed metabolic turnover in a substrate layer. The digital data are measured with a two-dimensional spectrophotometer based on a computerized video equipment with high spatial, temporal and intensity resolution. By application of three-dimensional procedures detailed structural properties of chemical and biochemical model systems will be presented yielding localization of reaction and transport events.