Dibucaine,chlorpromazine, and detergents mediate membrane breakdown in potato tuber homogenates

Various strategies were evaluated for their ability to minimize the rate of breakdown of endogenous membrane lipids during cell fractionation studies with potato tubers. Buffering the homogenates at pH 7.5 to 8.0 resulted in significantly lower rates of phosphatidylcholine (PC) hydrolysis than were observed at lower pH values. Several potential inhibitors were added to homogenates to evaluate their ability to inhibit membrane lipid hydrolysis. The addition of bovine serum albumin (1% w/v) inhibited the rate of PC hydrolysis by 50%. The addition of low concentrations (25–100 μM) dibucaine (nupercaine) inhibited PC hydrolysis, but at higher concentrations (1-2 mM) it caused a 5- to 6-fold stimulation. Because dibucaine is a calmodulin antagonist, two other calmodulin antagonists (trifluoperazine and chlorpromazine) were tested and found to exhibit similar patterns of inhibition and stimulation. Similarly, the addition of low concentrations of deoxycholate also inhibited PC hydrolysis and high concentrations stimulated it. These results indicate that the hydrophobic properties of deoxycholate, dibucaine, and other calmodülin antagonists may explain their unusual effects on the rates of PC hydrolysis in potato tuber homogenates. Although the addition of exogenous calcium increased the rate of PC hydrolysis, the addition of calmodulin (bovine brain) had no effect. Other experiments revealed that the addition of 1% bovine serum albumin improved the yield and stability of mitochondrial and microsomal fractions from potato tubers. In constrast, the addition of 100 μM dibucaine caused deleterious effects.     

Lotus corniculatus nodulation specificity is changed by the presence of a soybean lectin gene

Plant lectins have been implicated as playing an important role in mediating recognition and specificity in the Rhizobium-legume nitrogen-fixing symbiosis. To test this hypothesis, we introduced the soybean lectin gene Le1 either behind its own promoter or behind the cauliflower mosaic virus 35S promoter into Lotus corniculatus, which is nodulated by R. loti. We found that nodulelike outgrowths developed on transgenic L. corniculatus plant roots in response to Bradyrhizobium japonicum, which nodulates soybean and not Lotus spp. Soybean lectin was properly targeted to L. corniculatus root hairs, and although infection threads formed, they aborted in epidermal or hypodermal cells. Mutation of the lectin sugar binding site abolished infection thread formation and nodulation. Incubation of bradyrhizobia in the nodulation (nod) gene-inducing flavonoid genistein increased the number of nodulelike outgrowths on transgenic L. corniculatus roots. Studies of bacterial mutants, however, suggest that a component of the exopolysaccharide surface of B. japonicum, rather than Nod factor, is required for extension of host range to the transgenic L. corniculatus plants.     

All pyruvylated galactose in Schizosaccharomyces pombe N-glycans is present in the terminal disaccharide, 4, 6-O-[(R)-(1- carboxyethylidine)]-Galbeta1,3Galalpha1-

The large N-linked oligosaccharides released from Schizosaccharomyces pombe by endo-beta-N-acetylglucosaminidase H were examined to determine how the negatively chargedpyruvylated galactoses present (Gemmill,T.R., and Trimble,R.B., 1996, J. Biol. Chem ., 271, 25945-25949) were attached to the oligosaccharide chains. Binding of biotinylated human serum amyloid P and peanut agglutinin to native and depyruvylated S.pombe glycoproteins, respectively, indicated that the pyruvylated epitope was likely to be in the beta configuration. Examination by high- field 1H NMR of whole glycans and a disaccharide fragment released from them on partial acid hydrolysis showed that the pyruvylated galactose species was in fact beta1,3-linked to a second galactose, and this occurred an average of five to six times on nominal Gal57Man64GlcNAc N- glycans. The pyruvate-2,(4,6)Gal-beta1,3Gal epitope is chemically similar to acetaldehyde-Galbeta1,3Gal groups found on the glycoproteins from Paramyxovirus-infected bovine kidney cells (Prehm, P., Scheid,A. and Choppin,P.W. ,1979, J. Biol. Chem ., 254, 9669-9677). The 1:1 stoichiometry between pyruvate and beta-linked galactose in these S.pombe glycans indicates that either pyruvate addition to terminal beta1,3Gal is highly efficient or that pyruvylated Gal is transferred en bloc to alpha1,2-linked Gal residues in theN-linked chains. In contradiction to many galactomannan-producing fungi, which add substantial amounts of Gal in the furanose form to their glycoproteins, all detectable Gal in the large S.pombe galactomannans is in the pyranose form, as found in higher eukaryotes. The current work shows that the S.pombe outer chain structure is a poly-alpha1,6Man backbone 2- O-substituted with either Gal or the pyruvylated galactobiose and contains little alpha1,2-linked or 2-O-substituted Man. This is in contrast to the S. cerevisiae outer chain, which is poly-alpha1,6Man substituted with alpha1,2-linked Man sidechains (Ballou,C.E. ,1990, Methods Enzymol , 185, 440-470).      

Focus on RNA isolation: obtaining RNA for microRNA (miRNA) expression profiling analyses of neural tissue

MicroRNAs (miRNAs) are present in all known plant and animal tissues and appear to be somewhat concentrated in the mammalian nervous system. Many different miRNA expression profiling platforms have been described. However, relatively little research has been published to establish the importance of 'upstream' variables in RNA isolation for neural miRNA expression profiling. We tested whether apparent changes in miRNA expression profiles may be associated with tissue processing, RNA isolation techniques, or different cell types in the sample. RNA isolation was performed on a single brain sample using eight different RNA isolation methods, and results were correlated using a conventional miRNA microarray and then cross-referenced to Northern blots. Differing results were seen between samples obtained using different RNA isolation techniques and between microarray and Northern blot results. Another complication of miRNA microarrays is tissue-level heterogeneity of cellular composition. To investigate this phenomenon, miRNA expression profiles were determined and compared between highly-purified primary cerebral cortical cell preparations of rat primary E15-E18 neurons versus rat primary E15-E18 astrocytes. Finally, to assess the importance of dissecting human brain gray matter from subjacent white matter in cerebral cortical studies, miRNA expression profiles were compared between gray matter and immediately contiguous white matter. The results suggest that for microarray studies, cellular composition is important, and dissecting white matter from gray matter improves the specificity of the results. Based on these data, recommendations for miRNA expression profiling in neural tissues, and considerations worthy of further study, are discussed.     

Twenty-four-well plate miniature bioreactor high-throughput system: assessment for microbial cultivations

High-throughput (HT) miniature bioreactor (MBR) systems are becoming increasingly important to rapidly perform clonal selection, strain improvement screening, and culture media and process optimization. This study documents the initial assessment of a 24-well plate MBR system, Micro (micro)-24, for Saccharomyces cerevisiae, Escherichia coli, and Pichia pastoris cultivations. MBR batch cultivations for S. cerevisiae demonstrated comparable growth to a 20-L stirred tank bioreactor fermentation by off-line metabolite and biomass analyses. High inter-well reproducibility was observed for process parameters such as on-line temperature, pH and dissolved oxygen. E. coli and P. pastoris strains were also tested in this MBR system under conditions of rapidly increasing oxygen uptake rates (OUR) and at high cell densities, thus requiring the utilization of gas blending for dissolved oxygen and pH control. The E. coli batch fermentations challenged the dissolved oxygen and pH control loop as demonstrated by process excursions below the control set-point during the exponential growth phase on dextrose. For P. pastoris fermentations, the micro-24 was capable of controlling dissolved oxygen, pH, and temperature under batch and fed-batch conditions with subsequent substrate shot feeds and supported biomass levels of 278 g/L wet cell weight (wcw). The average oxygen mass transfer coefficient per non-sparged well were measured at 32.6 +/- 2.4, 46.5 +/- 4.6, 51.6 +/- 3.7, and 56.1 +/- 1.6 h(-1) at the operating conditions of 500, 600, 700, and 800 rpm shaking speed, respectively. The mixing times measured for the agitation settings 500 and 800 rpm were below 5 and 1 s, respectively.     

Cloning and gene map assignment of the Xiphophorus DNA ligase 1 gene

Fishes represent the stem vertebrate condition and have maintained several gene arrangements common to mammalian genomes throughout the 450 Myr of divergence from a common ancestor. One such syntenic arrangement includes the GPI-PEPD enzyme association on Xiphophorus linkage group IV and human chromosome 19. Previously we assigned the Xiphophorus homologue of the human ERCC2 gene to linkage group U5 in tight association with the CKM locus. CKM is also tightly linked to the ERCC2 locus on human chromosome 19, leading to speculation that human chromosome 19 may have arisen by fusion of two ancestral linkage groups which have been maintained in fishes. To investigate this hypothesis further, we isolated and sequenced Xiphophorus fish genomic regions exhibiting considerable sequence similarity to the human DNA ligase 1 amino acid sequence. Comparison of the fish DNA ligase sequence with those of other species suggests several modes of amino acid conservation in this gene. A 2.2-kb restriction fragment containing part of an X. maculatus DNA ligase 1 exon was used in backcross hybrid mapping with 12 enzyme or RFLP loci. Significant linkage was observed between the nucleoside phosphorylase (NP2) and the DNA ligase (LIG1) loci on Xiphophorus linkage group VI. This assignment suggests that the association of four DNA repair-related genes on human chromosome 19 may be the result of chance chromosomal rearrangements.      

Electro-optical behaviour of CuFe2O4@rGO nanocomposite for nonenzymatic detection of uric acid via the electrochemical method

Uric acid (UA) is a blood and urine component obtained as a metabolic by-product of purine nucleotides. Abnormalities in UA metabolism cause crystal deposition as monosodium urate and lead to various diseases such as gout, hyperuricemia, Lesch–Nyhan syndrome, etc. Monitoring these diseases requires a rapid, sensitive, selective, and portable detection approach. Therefore, this study demonstrates the hydrothermal synthesis of CuFe₂O₄/reduced graphene oxide (rGO) nanocomposite for selective detection of UA. After the nanocomposite synthesis, characterization was performed by X-ray diffraction spectroscopy, Fourier transform infrared spectroscopy, Raman spectroscopy, X-ray photoelectron spectroscopy, UV–visible spectrometry, atomic force spectroscopy, scanning electron microscopy, and electrochemical analysis. Furthermore, from the electrochemical analysis using cyclic voltammetry (CV), kinetic studies were carried out by varying the scan rate to obtain the diffusion coefficient, surface concentration, and rate of charge transfer to achieve a calibration curve that indicates the quasi reversible nature of the fabricated electrode with a linear regression coefficient of oxidation (R²: 0.9992) and reduction (R²: 0.9971) peaks. Moreover, the fabricated nonenzymatic amperometric sensor to detect UA with a linearity (R²: 0.9989) of 1–400 μM was highly sensitive (2.75 × 10⁻⁴ mAμM⁻¹ cm⁻²) and had a lower limit of detection (0.01231 μM) at pH 7.5 in phosphate-buffered saline solution. Therefore, the CuFe₂O₄/rGO/ITO-based nonenzymatic sensor could detect interfering agents and spiked real bovine serum samples with higher sensitivity and selectivity for UA detection.     

An RNA folding method capable of identifying pseudoknots and base triples

MOTIVATION: Recently, we described a Maximum Weighted Matching (MWM) method for RNA structure prediction. The MWM method is capable of detecting pseudoknots and other tertiary base-pairing interactions in a computationally efficient manner (Cary and Stormo, Proceedings of the Third International Conference on Intelligent Systems for Molecular Biology, pp. 75-80, 1995). Here we report on the results of our efforts to improve the MWM method's predictive accuracy, and show how the method can be extended to detect base interactions formerly inaccessible to automated RNA modeling techniques. RESULTS: Improved performance in MWM structure prediction was achieved in two ways. First, new ways of calculating base pair likelihoods have been developed. These allow experimental data and combined statistical and thermodynamic information to be used by the program. Second, accuracy was improved by developing techniques for filtering out spurious base pairs predicted by the MWM program. We also demonstrate here a means by which the MWM folding method may be used to detect the presence of base triples in RNAs. AVAILABILITY: http://www.cshl.org/mzhanglab/tabaska/j axpage. html CONTACT: tabaska@cshl.org