The effect of acridine orange on the structure of ribosomes

Exposure of Escherichia coli MRE-600 ribosomes to acridine orange (AO) at low ionic strength (1mM Tris-acetate pH 7.4) results in quantitative binding of the dye. Under our experimental conditions about a few hundred dye molecules can be bound to any one of the 30, 50, or 70-S particles. AO causes the 30 and the 50-S subunits to form ribosomal aggregates of approximate sedimentation constants of 70 and 100-S.     

Changes in avoidance behaviour following ethanol treatment in rats of different ages

Avoidance learning and pain sensitivity were studied in rats after chronic ethanol administration (1.1 to 1.7 g per kg b.w. in drinking water) at prenatal, adolescent and adult ages. The behavioural reactions were tested in adulthood by studying passive and active avoidance learning and the threshold of pain sensitivity to electric tail shock. Chronic ethanol consumption led to an impairment of avoidance learning and to hyperalgesia in each experimental series, although the alterations were greater in the prenatally treated groups. The experimental observations are discussed in the light of fetal alcohol syndrome.     

Impact of fungicides on Metarhizium anisopliae in the rhizosphere, bulk soil and in vitro

The entomopathogenic fungus Metarhizium anisopliae (Metchnikoff) Sorokin (Hypocreales: Clavicipitaceae) is registered in the United States and The Netherlands for black vine weevil, Otiorhynchus sulcatus (Coleoptera: Curculionidae) control in container-grown ornamentals. These studies were conducted to determine the compatibility of M. anisopliae (F52) with a wide range of fungicides commonly applied to container-grown ornamentals for the management of soil-borne plant pathogens. The impact of fungicides on spore germination and mycelial growth were determined in vitro. In addition, M. anisopliae persistence in bulk and rhizosphere soil was determined 30 days following dual application of each fungicide at 7–28 days intervals as prescribed. A number of fungicides (thiophanate-methyl, dimethomorph, captan, triflumizole, triflozystrobin, pyraclostrobin, azoxystrobin) inhibited spore germination in vitro. A larger number of fungicides (fosetyl-AI, thiophanate-methyl, dimethomorph, captan, quintozene, triflumizole, fludioxanil, triflozystrobin, pyraclostrobin, fludiox-mefanox, iprodione, azoxystrobin, phosphorus acid/K-salts) inhibited mycelial growth in vitro. Only three fungicides (etridiazole, propamocard and mafanoxam) had no significant impact in vitro on spore germination or mycelial growth. While a number of fungicides had a detrimental impact in vitro, there was no impact on M. anisopliae populations in bulk soil following dual application of any fungicide. However, the fungicides captan and triflumizolet, which have a short reapplication interval, had a detrimental impact on M. anisopliae populations in the rhizosphere. As researchers develop rhizosphere competence as an alternative management strategy for black vine weevil, the fungicides captan and triflumizole should be avoided.     

Differential regulation of translation and endocytosis of alternatively spliced forms of the type II bone morphogenetic protein (BMP) receptor

The expression and function of transforming growth factor-β superfamily receptors are regulated by multiple molecular mechanisms. The type II BMP receptor (BMPRII) is expressed as two alternatively spliced forms, a long and a short form (BMPRII-LF and –SF, respectively), which differ by an ∼500 amino acid C-terminal extension, unique among TGF-β superfamily receptors. Whereas this extension was proposed to modulate BMPRII signaling output, its contribution to the regulation of receptor expression was not addressed. To map regulatory determinants of BMPRII expression, we compared synthesis, degradation, distribution, and endocytic trafficking of BMPRII isoforms and mutants. We identified translational regulation of BMPRII expression and the contribution of a 3’ terminal coding sequence to this process. BMPRII-LF and -SF differed also in their steady-state levels, kinetics of degradation, intracellular distribution, and internalization rates. A single dileucine signal in the C-terminal extension of BMPRII-LF accounted for its faster clathrin-mediated endocytosis relative to BMPRII-SF, accompanied by mildly faster degradation. Higher expression of BMPRII-SF at the plasma membrane resulted in enhanced activation of Smad signaling, stressing the potential importance of the multilayered regulation of BMPRII expression at the plasma membrane.     

A Gene-Specific Method for Predicting Hemophilia-Causing Point Mutations

A fundamental goal of medical genetics is the accurate prediction of genotype–phenotype correlations. As an approach to develop more accurate in silico tools for prediction of disease-causing mutations of structural proteins, we present a gene- and disease-specific prediction tool based on a large systematic analysis of missense mutations from hemophilia A (HA) patients. Our HA-specific prediction tool, HApredictor, showed disease prediction accuracy comparable to other publicly available prediction software. In contrast to those methods, its performance is not limited to non-synonymous mutations. Given the role of synonymous mutations in disease and drug codon optimization, we propose that utilizing a gene- and disease-specific method can be highly useful to make functional predictions possible even for synonymous mutations. Incorporating computational metrics at both nucleotide and amino acid levels along with multiple protein sequence/structure alignment significantly improved the predictive performance of our tool. HApredictor is freely available for download at http://www.ncbi.nlm.nih.gov/CBBresearch/Przytycka/HA_Predict/index.htm.     

Small molecules restore the function of mutant CLC5 associated with Dent disease

Dent disease type 1 is caused by mutations in the CLCN5 gene that encodes CLC5, a 2Cl⁻/H⁺ exchanger. The CLC5 mutants that have been functionally analysed constitute three major classes based on protein expression, cellular localization and channel function. We tested two small molecules, 4-phenylbutyrate (4PBA) and its analogue 2-naphthoxyacetic acid (2-NOAA), for their effect on mutant CLC5 function and expression by whole-cell patch-clamp and Western blot, respectively. The expression and function of non-Class I CLC5 mutants that have reduced function could be restored by either treatment. Cell viability was reduced in cells treated with 2-NOAA. 4PBA is a FDA-approved drug for the treatment of urea cycle disorders and offers a potential therapy for Dent disease.     

Abnormal stomatal behavior and root resistance,and hormonal imbalance in three wilty mutants of tomato

Three wilty mutants of tomato, flacca, sitiens, and notabilis, were compared with two control normal cultivars, Rheinlands Ruhm and Lukullus, for concentration of abscisic acid and root resistance to water flow. In addition, the three mutants treated with abscisic acid during development were compared with control mutant plants for stomatal opening and root resistance. The hormonal concentration was estimated by coleoptile assay and gas chromatography. Stomatal opening was estimated by measuring rate of transpiration and by examining leaf imprints. Root resistance was estimated by measuring the amount of exudate from roots of decapitated plants and the difference between the osmotic pressure of the exudate and the root medium. A lower level of abscisic acid was found in all three mutants as compared with the control normal plants. In addition, root resistance to water flow was higher in the three mutants than in the control normal types. All three mutants completely reverted to normal phenotypically, including stomatal and root resistances, when treated with abscisic acid. It has been suggested that the first hormonal change in the mutants is that of abscisic acid, and from it proceed the other changes.This work was supported in part by a research grant from the Ford Foundation (Ford-6, A-VII).     

Enzymes concerned with beta-carboxylation in marine phytoplankter. Purification and properties of phosphoenolpyruvate carboxykinase.

The enzyme resonsible for β-carboxylation, with eventual incorporation of CO₂ into amino acids, has been studied in three diatoms and a dinoflagellate. The enzyme from Phaeodactylum tricornutum has been purified to homogeneity and has an absolute requirement for ADP and Mn²⁺. This enzyme is best described as phosphoenolpyruvate carboxykinase (carboxylating), but it differs considerably from enzymes with the same name, isolated from mammalian and bacterial sources, in that the reaction of phosphoenolpyruvate with bicarbonate lies strongly in the direction of formation of oxaloacetate and ATP. This conservation of the high-energy phosphate groups of phosphoenolpyruvate in ATP would be advantageous to diatoms living at low levels of light intensity in seawater.