Within-species correlations in leaf traits of three boreal plant species along a latitudinal gradient

Evergreen boreal plant species express high variability in their leaf traits. It remains controversial whether this within-species variability is constrained to the same leaf trait relationships as has been observed across species. We sampled leaves of three boreal evergreen woody species along a latitudinal gradient (from 57o56′N to 69o55′N). Leaf longevity (LL) of Pinus sylvestris L. and Vaccinium vitis-idaea L. correlated negatively with mean annual air temperature (MAT), whereas the LL of Ledum palustre L. was not affected by MAT. V. vitis-idaea and L. palustre had a negative relationship between leaf mass per area (LMA) and MAT. In P. sylvestris, the LMA–MAT relationship was positive. A negative correlation between LL and LMA was significant only for P. sylvestris. Leaf nitrogen concentration was positively related to leaf phosphorus concentration in all three species. Leaf potassium concentration was related to nitrogen concentration only in L. palustre, and to phosphorus concentration in P. sylvestris and L. palustre. Our results demonstrate that although within the studied species the variation in some of the leaf traits may have the same degree as interspecific variation, there is no such intercorrelation of leaf traits within the studied species as has been observed across species.     

Gene order of the TOL catabolic plasmid upper pathway operon and oxidation of both toluene and benzyl alcohol by the xylA product.

TOL plasmid pWW0 specifies enzymes for the oxidative catabolism of toluene and xylenes. The upper pathway converts the aromatic hydrocarbons to aromatic carboxylic acids via corresponding alcohols and aldehydes and involves three enzymes: xylene oxygenase, benzyl alcohol dehydrogenase, and benzaldehyde dehydrogenase. The synthesis of these enzymes is positively regulated by the product of xylR. Determination of upper pathway enzyme levels in bacteria carrying Tn5 insertion mutant derivatives of plasmid pWW0-161 has shown that the genes for upper pathway enzymes are organized in an operon with the following order: promoter-xylC (benzaldehyde dehydrogenase gene[s])-xylA (xylene oxygenase gene[s])-xylB (benzyl alcohol dehydrogenase gene). Subcloning of the upper pathway genes in a lambda pL promoter-containing vector and analysis of their expression in Escherichia coli K-12 confirmed this order. Two distinct enzymes were found to attack benzyl alcohol, namely, xylene oxygenase and benzyl alcohol dehydrogenase; and their catalytic activities were additive in the conversion of benzyl alcohol to benzaldehyde. The fact that benzyl alcohol is both a product and a substrate of xylene oxygenase indicates that this enzyme has a relaxed substrate specificity.     

DNA binding of Jun and Fos bZip domains: homodimers and heterodimers induce a DNA conformational change in solution.

We constructed plasmids encoding the sequences for the bZip modules of c-Jun and c-Fos which could then be expressed as soluble proteins in Escherichia coli. The purified bZip modules were tested for their binding capacities of synthetic oligonucleotides containing either TRE or CRE recognition sites in electrophoretic mobility shift assays and circular dichroism (CD). Electrophoretic mobility shift assays showed that bZip Jun homodimers and bZip Jun/Fos heterodimers bind a collagenase-like TRE (CTGACTCAT) with dissociation constants of respectively 1.4 x 10(-7) M and 5 x 10(-8) M. As reported earlier [Patel et al. (1990) Nature 347, 572-575], DNA binding induces a marked change of the protein structure. However, we found that the DNA also undergoes a conformational change. This is most clearly seen with small oligonucleotides of 13 or 14 bp harboring respectively a TRE (TGACTCA) or a CRE (TGACGTCA) sequence. In this case, the positive DNA CD signal at 280 nm increases almost two-fold with a concomitant blue-shift of 3-4 nm. Within experimental error the same spectral changes are observed for TRE and CRE containing DNA fragments. The spectral changes observed with a non-specific DNA fragment are weaker and the signal of free DNA is recovered upon addition of much smaller salt concentrations than required for a specific DNA fragment. Surprisingly the spectral changes induced by Jun/Jun homodimers are not identical to those induced by Jun/Fos heterodimers. However, in both cases the increase of the positive CD band and the concomitant blue shift would be compatible with a B to A-transition of part of the binding site or a DNA conformation intermediate between the canonical A and B structures.     

Aerobic catabolism of bile acids.

Seventy-eight stable cultures obtained by enrichment on media containing ox bile or a single bile acid were able to utilize one or more bile acids, as well as components of ox bile, as primary carbon sources for growth. All isolates were obligate aerobes, and most (70) were typical (48) or atypical (22) Pseudomonas strains, the remainder (8) being gram-positive actinomycetes. Of six Pseudomonas isolates selected for further study, five produced predominantly acidic catabolites after growth on glycocholic acid, but the sixth, Pseudomonas sp. ATCC 31752, accumulated as the principal product a neutral steroid catabolite. Optimum growth of Pseudomonas sp. ATCC 31752 on ox bile occurred at pH 7 to 8 and from 25 to 30 degrees C. No additional nutrients were required to sustain good growth, but growth was stimulated by the addition of ammonium sulfate and yeast extract. Good growth was obtained with a bile solids content of 40 g/liter in shaken flasks. A near-theoretical yield of neutral steroid catabolites, comprising a major (greater than 50%) and three minor products, was obtained from fermentor growth of ATCC 31752 in 6.7 g of ox bile solids per liter. The possible commercial exploitation of these findings to produce steroid drug intermediates for the pharmaceutical industry is discussed.     

Deoxycholic acid degradation by a Pseudomonas species. Acidic intermediates from the initial part of the catabolic pathway.

The microbial catabolism of deoxycholic acid by a Pseudomonas species was studied, and three acidic products were isolated as their methyl esters. Evidence is presented that the compounds are methyl 3 alpha,12 alpha-dihydroxy-23,24-dinor-5 beta-cholan-22-oate, methyl 12 alpha-hydroxy-3-oxo-5 beta-cholan-24-oate and methyl 12 alpha-hydroxy-3-oxo-23,24-dinor-5 beta-cholan-22-oate.     

Dilation of human atria: Increased diffusion restrictions for ADP,overexpression of hexokinase 2 and its coupling to oxidative phosphorylation in cardiomyocytes

Cardiac energy metabolism with emphasis on mitochondria was addressed in atrial tissue from patients with overload-induced atrial dilation. Structural remodeling of dilated (D) atria manifested as intracellular accumulation of fibrillar aggregates, lipofuscin, signs of myolysis and autophagy. Despite impaired complex I dependent respiration and increased diffusion restriction for ADP, no changes regarding adenylate and creatine kinase occurred. We observed 7-fold overexpression of HK2 gene in D atria with concomitant 2-fold greater activation of mitochondrial oxygen consumption by glucose, which might represent an adaption to increased energy requirements and impaired mitochondrial function by effectively joining glycolysis and oxidative phosphorylation.     

Substrate specificity of the pseudouridine synthase RluD in Escherichia coli

Pseudouridine synthase RluD converts uridines at positions 1911, 1915, and 1917 of 23S rRNA to pseudouridines. These nucleotides are located in the functionally important helix-loop 69 of 23S rRNA. RluD is the only pseudouridine synthase that is required for normal growth in Escherichia coli. We have analyzed substrate specificity of RluD in vivo. Mutational analyses have revealed: (a) RluD isomerizes uridine in vivo only at positions 1911, 1915, and 1917, regardless of the presence of uridine at other positions in the loop of helix 69 of 23S rRNA variants; (b) substitution of one U by C has no effect on the conversion of others (i.e. formation of pseudouridines at positions 1911, 1915, and 1917 are independent of each other); (c) A1916 is the only position in the loop of helix 69, where mutations affect the RluD specific pseudouridine formation. Pseudouridines were determined in the ribosomal particles from a ribosomal large subunit defective strain (RNA helicase DeaD(-)). An absence of pseudouridines in the assembly precursor particles suggests that RluD directed isomerization of uridines occurs as a late step during the assembly of the large ribosomal subunit.     

Mutational analysis of Aedes aegypti Dicer 2 provides insights into the biogenesis of antiviral exogenous small interfering RNAs

The exogenous small interfering RNA (exo-siRNA) pathway is a key antiviral mechanism in the Aedes aegypti mosquito, a widely distributed vector of human-pathogenic arboviruses. This pathway is induced by virus-derived double-stranded RNAs (dsRNA) that are cleaved by the ribonuclease Dicer 2 (Dcr2) into predominantly 21 nucleotide (nt) virus-derived small interfering RNAs (vsiRNAs). These vsiRNAs are used by the effector protein Argonaute 2 within the RNA-induced silencing complex to cleave target viral RNA. Dcr2 contains several domains crucial for its activities, including helicase and RNase III domains. In Drosophila melanogaster Dcr2, the helicase domain has been associated with binding to dsRNA with blunt-ended termini and a processive siRNA production mechanism, while the platform-PAZ domains bind dsRNA with 3’ overhangs and subsequent distributive siRNA production. Here we analyzed the contributions of the helicase and RNase III domains in Ae. aegypti Dcr2 to antiviral activity and to the exo-siRNA pathway. Conserved amino acids in the helicase and RNase III domains were identified to investigate Dcr2 antiviral activity in an Ae. aegypti-derived Dcr2 knockout cell line by reporter assays and infection with mosquito-borne Semliki Forest virus (Togaviridae, Alphavirus). Functionally relevant amino acids were found to be conserved in haplotype Dcr2 sequences from field-derived Ae. aegypti across different continents. The helicase and RNase III domains were critical for silencing activity and 21 nt vsiRNA production, with RNase III domain activity alone determined to be insufficient for antiviral activity. Analysis of 21 nt vsiRNA sequences (produced by functional Dcr2) to assess the distribution and phasing along the viral genome revealed diverse yet highly consistent vsiRNA pools, with predominantly short or long sequence overlaps including 19 nt overlaps (the latter representing most likely true Dcr2 cleavage products). Combined with the importance of the Dcr2 helicase domain, this suggests that the majority of 21 nt vsiRNAs originate by processive cleavage. This study sheds new light on Ae. aegypti Dcr2 functions and properties in this important arbovirus vector species.     

Studies of mitochondrial respiration in muscle cells in situ: Use and misuse of experimental evidence in mathematical modelling

Applications of permeabilized cell and skinned fiber techniques in combination with methods of mathematical modelling for studies of mitochondrial function in the cell are critically evaluated. Mathematical models may be useful tools for explaining biological phenomena, but only if they are selected by fitting the computing results with real experimental data. Confocal microscopy has been used in experiments with permeabilized cardiomyocytes and myocardial fibers to determine the maximal diffusion distance from medium to the core of cells, which is shown not to exceed 8-10 microm. This is a principal index for correctly explaining high apparent Km for exogenous ADP (200-300 microM) in regulation of mitochondrial respiration in oxidative muscle cells in situ. The best fitting of the results of in silico studies may be achieved by using of the compartmentalized energy transfer model. From these results, it may be concluded that in cardiac muscle cells the mitochondria and ATPases are organized into intracellular energetic units (ICEUs) separated from the bulk phase of cytoplasm by some barriers which limit the diffusion of adenine nucleotides. In contrast, alternative models based on the concept of the cell as homogenous system do not explain the observed experimental phenomena and have led to misleading conclusions. The various sources of experimental and conceptual errors are analyzed.