Decreased Long-Chain Fatty Acyl CoA Elongation Activity in Quaking and Jimpy Mouse Brain: Deficiency in One Enzyme or Multiple Enzyme Activities?

Using long-chain fatty acyl CoAs (arachidoyl CoA and behenoyl CoA), a decrease in overall fatty acid chain elongation activity was observed in the quaking and jimpy mouse brain microsomes relative to controls. Arachidoyl CoA (20:0) and behenoyl CoA (22:0) elongation activities were depressed to about 50% and 80% of control values in quaking and jimpy mice, respectively. Measurement of the individual enzymatic activities of the elongation system revealed a single deficiency in enzyme activity; only the condensation activity was reduced to the same extent as total elongation in both quaking and jimpy mice. The activities of the other three enzymes, beta-ketoacyl CoA reductase, beta-hydroxyacyl CoA dehydrase, and trans-2-enoyl CoA reductase, in both mutants were similar to the activities present in the control mouse. In addition, the activities of these three enzymes were more than two to three orders of magnitude greater than the condensing enzyme activity in all three groups, establishing that the condensing enzyme catalyzes the rate-limiting reaction step of total elongation. When the elongation of palmitoyl CoA was measured, only a 25% decrease in total elongation occurred in both mutants; a similar percent decrease in the condensation of palmitoyl CoA also was observed. The activities of the other three enzymes were unaffected. These results support the concept of either multiple elongation pathways or multiple condensing enzymes.     

An Enzyme from Aristolochia indica Destabilizes Fibrin-β Amyloid Co-Aggregate: Implication in Cerebrovascular Diseases

Fibrinogen and β-amyloid (Aβ) peptide independently form ordered aggregates but in combination, they form disordered structures which are resistant to fibrinolytic enzymes like plasmin and cause severity in cerebral amyloid angiopathy (CAA). A novel enzyme of 31.3 kDa has been isolated from the root of the medicinal plant Aristolochia indica that showed fibrinolytic as well as fibrin-Aβ co-aggregate destabilizing properties. This enzyme is functionally distinct from plasmin. Thrombolytic action of the enzyme was demonstrated in rat model. The potency of the plant enzyme in degrading fibrin and fibrin-plasma protein (Aβ, human serum albumin, lysozyme, transthyretin and fibronectin) co-aggregates was demonstrated by atomic force microscopy, scanning electron microscopy and confocal microscopy that showed better potency of the plant enzyme as compared to plasmin. Moreover, the plant enzyme inhibited localization of the co-aggregate inside SH-SY5Y human neuroblastoma cells and also co-aggregate induced cytotoxicity. Plasmin was inefficient in this respect. In the background of limited options for fragmentation of these co-aggregates, the plant enzyme may appear as a potential proteolytic enzyme.     

Characterization of Brain β-Carboline-2-N-Methyltransferase,An Enzyme That May Play a Role in Idiopathic Parkinson's Disease

The activity of -carboline-2-N-methyltransferase results in the formation of neurotoxic N-methylated -carbolinium compounds. We have hypothesized that these N-methylated -carbolinium cations may contribute to the development of idiopathic Parkinson's disease. This report describes experiments undertaken to optimize assay conditions for bovine brain -carboline-2-N-methyltransferase activity. The activity of -carboline-2-N-methyltransferase is primarily localized in the cytosol, has a pH optimum of 8.5–9, and obeys Michaelis-Menten kinetics with respect to its substrates, 9-methylnorharman (9-MeNH) and S-adenosyl-L-methionine (SAM). Kinetic constants, K_M and V_max, with respect to 9-MeNH, are 75 M and 48 pmol/h/mg protein, respectively. The K_M for SAM is 81 M and the V_max is 53 pmol/h/mg protein. In addition, enzyme activity is inhibited by S-adenosyl-L-homocysteine (SAH) or zinc, and is increased 2-fold in the presence of iron or manganese. Enzyme characterization is a prerequisite to the purification of this N-methyltransferase from bovine brain as well as comparison of its activity in human brain from control and Parkinson's disease individuals.     

Acyl coenzyme A: Phospholipid acyltransferases in porcine platelets discriminate between ω-3 and ω-6 unsaturated fatty acids

The properties of porcine platelet acyltransferases which catalyze the incorporation of unsaturated fatty acids into the 2 positions of phospholipids were compared with those of porcine liver microsomes and rat liver microsomes. There were significant differences in the relative rates of incorporation of acyl groups into phospholipids as catalyzed by the membranes from different species and organs. The 1-acylglycerophosphate acyltransferase system showed relatively broad specificity for saturated and unsaturated fatty acids, with 14- to 20-carbon chains, while unsaturated acyl-CoAs with 18- and 20-carbon chains were generally good substrates in the acylations of 1-acylglycerophosphocholine and 1-acylglycerophosphoinositol. ω-3 and ω-6 unsaturated fatty acids were recognized differently by different acyltransferase systems in platelets. When activities for combinations of ω-3 and ω-6 unsaturated acyl-CoAs with the same number of carbons and with similar number of double bonds were compared, ω-6 fatty acids were relatively more preferred substrates than ω-3 fatty acids for the 1-acylglycerophosphoinositol acyltransferase system as compared with 1-acylglycerophosphocholine acyltransferase system.     

Nitrous Oxide Reduction in Nodules: Denitrification or N2 Fixation?

Detached cowpea nodules that contained a nitrous oxide reductase-positive (Nor⁺) rhizobium strain (8A55) and a nitrous oxide reductase-negative (Nor⁻) rhizobium strain (32H1) were incubated with 1% ¹⁵N₂O (95 atom% ¹⁵N) in the following three atmospheres: (i) aerobic with C₂H₂ (10%), (ii) aerobic without C₂H₂, and (iii) anaerobic (argon atmosphere) without C₂H₂. The greatest production of ¹⁵N₂ occurred anaerobically with 8A55, yet very little was formed with 32H1. Although acetylene reduction activity was slightly higher with 32H1, about 10 times more ¹⁵N₂ was produced aerobically by 8A55 than by 32H1 in the absence of acetylene. The major reductive pathway of N₂O reduction by denitrifying rhizobium strain 8A55 is by nitrous oxide reductase rather than nitrogenase.     

Δ′-Pyrroline: An intermediary metabolite in the conversion of putrescine to 2-pyrrolidone

Δ′-Pyrroline, an oxidative product of putrescine metabolism, was chemically synthesized and incubated with a rat liver homogenate. The incubation mixture was fractionated on an amino acid analyzer before and after acid hydrolysis. The hydrolyzed sample, in contrast to the unhydrolyzed sample, contained a ninhydrin positive compound that cochromatographed with γ-aminobutyric acid, the product of 2-pyrrolidone acid hydrolysis. Authentic 2-pyrrolidone had the same retention time as the Δ′-pyrroline metabolite when analyzed by high-pressure liquid chromatography. It is concluded that Δ′-pyrroline is an intermediary metabolite in the pathway from putrescine to 5-hydroxy-2-pyrrolidone.     

An assessment of soil enrichment by actinorhizal N2 fixation using δ15N values in a chronosequence of deglaciation at Glacier Bay,Alaska

The extent of transfer of fixed N between N₂-fixing and non-N₂-fixing plant species is largely unknown in successional studies. In order to redress this deficiency at a locale intensively studied ecologically, leaf tissue samples were collected from actinorhizal N₂-fixing (Alnus, Shepherdia, and Dryas) and two non-N₂-fixing (Salix) woody species within research plots located along a chronosequence of deglaciated fjord in Glacier Bay National Park, Alaska. The tissue samples were analyzed for ¹⁵N content, and the resulting data analyzed for trends in plant tissue N. Among the non-N₂-fixing Salix species, ¹⁵N values increased from the most recently deglaciated sites to converge with the temporally more-stable values for the symbiotic N₂-fixing species on sites at about 40 years after deglaciation. The lower ¹⁵N values of sequestered N in plant tissues suggested that N derived from N₂-fixing plants accounts for the major portion of N in associated plants up to 40 years after deglaciation. The ¹⁵N isotopic data also suggested that Shepherdia canadensis depends least on soil N, D. drummondii the most, and A. viridis ssp. sinuata somewhere between those two species. The presence of a sere dominated by dense thickets of A. viridis ssp. sinuata at the convergence of ¹⁵N values for the N₂-fixing and non-N₂-fixing species indicated that this species is most responsible for accumulation of fixed N in soil at Glacier Bay. This paper is dedicated to the memory of Steven J. Kohls who died prior to publication of this research.     

An Efficient Method for the Synthesis of [4–15N]Cytidine, 2′-Deoxy[4–15N]Cytidine, [6–15N]adenosine,and 2′-Deoxy [6–15N]adenosine Derivatives

Abstract

Nucleophilic substitution reactions of 4-azolyl-1 β-P-D-ribofuranosylpyrimidin-2(1H)-one and 6-azolyl-9-β-D-ribofuranosyl-9H-purine derivatives, which were converted from uridine and inosine, with [¹⁵N]phthalimide in the presence of triethylamine or DBU gave N ⁴-phthaloyl[4-¹⁵N]cytidine and N ⁶-phthaloyl[6-¹⁵N]- adenosine derivatives, respectively, in high yields. Similar reactions of those azolyl derivatives with succinimide afforded N ⁴-succinylcytidine and N ⁶-succinyladenosine derivatives in high yields. The corresponding 2′-deoxyribonucleosides were also synthesized efficiently through the same procedure.

     

Rhamnogalacturonan α-d-Galactopyranosyluronohydrolase : An Enzyme That Specifically Removes the Terminal Nonreducing Galacturonosyl Residue in Rhamnogalacturonan Regions of Pectin1

A new enzyme, rhamnogalacturonan (RG) α-d-galactopyranosyluronohydrolase (RG-galacturonohydrolase), able to release a galacturonic acid residue from the nonreducing end of RG chains but not from homogalacturonan, was purified from an Aspergillus aculeatus enzyme preparation. RG-galacturonohydrolase acted with inversion of anomeric configuration, initially releasing β-d-galactopyranosyluronic acid. The enzyme cleaved smaller RG substrates with the highest catalytic efficiency. A Michaelis constant of 85 μm and a maximum reaction rate of 160 units mg⁻¹ was found toward a linear RG fragment with a degree of polymerization of 6. RG-galacturonohydrolase had a molecular mass of 66 kD, an isoelectric point of 5.12, a pH optimum of 4.0, and a temperature optimum of 50°C. The enzyme was most stable between pH 3.0 and 6.0 (for 24 h at 40°C) and up to 60°C (for 3 h).     

An alternate pathway to long-chain polyunsaturates: the FADS2 gene product ��8-desaturates 20:2n-6 and 20:3n-3

The mammalian Δ6-desaturase coded by fatty acid desaturase 2 (FADS2; HSA11q12-q13.1) catalyzes the first and rate-limiting step for the biosynthesis of long-chain polyunsaturated fatty acids. FADS2 is known to act on at least five substrates, and we hypothesized that the FADS2 gene product would have Δ8-desaturase activity. Saccharomyces cerevisiae transformed with a FADS2 construct from baboon neonate liver cDNA gained the function to desaturate 11,14-eicosadienoic acid (20:2n-6) and 11,14,17-eicosatrienoic acid (20:3n-3) to yield 20:3n-6 and 20:4n-3, respectively. Competition experiments indicate that Δ8-desaturation favors activity toward 20:3n-3 over 20:2n-6 by 3-fold. Similar experiments show that Δ6-desaturase activity is favored over Δ8-desaturase activity by 7-fold and 23-fold for n-6 (18:2n-6 vs 20:2n-6) and n-3 (18:3n-3 vs 20:3n-3), respectively. In mammals, 20:3n-6 is the immediate precursor of prostaglandin E1 and thromboxane B1. 20:3n-6 and 20:4n-3 are also immediate precursors of long-chain polyunsaturated fatty acids arachidonic acid and eicosapentaenoic acid, respectively. These findings provide unequivocal molecular evidence for a novel alternative biosynthetic route to long-chain polyunsaturated fatty acids in mammals from substrates previously considered to be dead-end products.     

Modeling 2hJiso(N, N) in nucleic acid base pairs: Ab initio characterization of the 2hJ(N, N) tensor in the methyleneimine dimer as a function of hydrogen bond geometry

The observation of ^2h J _iso(N, N) coupling has prompted considerable interest in this phenomenon from experimentalists and theoreticians due to the potential these couplings hold for the determination of secondary and tertiary structure in biologically important molecules. Here, we present an ab initio (MCSCF) study of the complete ^2h J(N, N) tensor for a model methyleneimine dimer system as a function of (i) the N-N separation, r _NN, and (ii) the hydrogen bond angle, . This simple system models the ^2h J(N, N) tensor of nucleic acid base pairs. Results indicate that although the Fermi-contact mechanism dominates ^2h J _iso(N, N), the coupling tensor is anisotropic due to contributions from the Fermi-contact spin-dipolar cross term. The variation in ^2h J _iso(N, N) as a function of r _NN is fit to an exponential decay. The influence of on the coupling constant is less pronounced but must be considered if experimental coupling constants are to be used for quantitative structure determination. Our results for this simple model system demonstrate that ^2h J _iso(N, N) is a valuable probe of hydrogen bonding in nucleic acid base pairs.     

4-(2′-Carboxyphenyl)-4-oxobutyrate: An obligatory intermediate in lawsone biosynthesis

4-(2′-Carboxyphenyl)-4-oxobutyric acid (6) has been detected in cuttings of Impatiens balsamina. It is labelled under conditions where activity from U-¹⁴C-glutamate is incorporated effectively into lawsone (1). 3-(2′-Carboxyphenyl)-3-oxopropionic acid (7) has also been encountered in the cuttings.     

An investigation into lanthanide–lanthanide magnetic interactions in a series of [Ln2(mdeaH2)2(piv)6] dimers

A series of six isostructural lanthanide dimers of general formula [Ln₂(mdeaH₂)₂(piv)₆], where mdeaH₂ is N-methyldiethanolamine, piv is pivalate, and Ln = La, Ce, Pr, Nd, Sm, and Gd, has been surveyed to gauge the nature of the magnetic interactions between the lanthanide centres. Single-crystal X-ray structure analyses indicate that the lanthanides are connected by syn,syn-carboxylate bridges. It was found from an analysis of their bulk magnetic susceptibilities as a function of temperature that this type of bridge mediates vanishingly small magnetic interactions. This finding is important in the context of developing synthetic strategies for the preparation of new molecular based magnetic materials.     

Association of MicroRNA-196a2 Variant with Response to Short-Acting β2-Agonist in COPD: An Egyptian Pilot Study

Chronic obstructive pulmonary disease (COPD) is a multifactorial chronic respiratory disease, characterized by an obstructive pattern. Understanding the genetic predisposition of COPD is essential to develop personalized treatment regimens. MicroRNAs (miRNAs) are small, endogenous, non-coding RNAs that modulate the expression levels of specific proteins based on sequence complementarity with their target mRNA molecules. Emerging evidences demonstrated the potential use of miRNAs as a disease biomarker. This pilot study aimed to investigate the association of the MIR-196a2 rs11614913 (C/T) polymorphism with COPD susceptibility, the clinical outcome and bronchodilator response to short-acting β₂-agonist. Genotyping of rs11614913 polymorphism was determined in 108 COPD male patients and 116 unrelated controls using real-time polymerase chain reaction technology. In silico target prediction and network core analysis were performed. COPD patients did not show significant differences in the genotype distribution (p = 0.415) and allele frequencies (p = 0.306) of the studied miRNA when compared with controls. There were also no associations with GOLD stage, dyspnea grade, disease exacerbations, COPD assessment test for estimating impact on health status score, or the frequency of intensive care unit admission. However, COPD patients with CC genotype corresponded to the smallest bronchodilator response after Salbutamol inhalation, the heterozygotes (CT) had an intermediate response, while those with the TT genotype showed the highest response (p < 0.001). In conclusion MIR-196a2 rs11614913 polymorphism is associated with the bronchodilator response of COPD in our sample of the Egyptian population, generating hypothesis of the potential use of MIR-196a2 variant as a pharmacogenetic marker for COPD.     

H5N2 avian influenza outbreak in Texas in 2004: the first highly pathogenic strain in the United States in 20 years?

In early 2004, an H5N2 avian influenza virus (AIV) that met the molecular criteria for classification as a highly pathogenic AIV was isolated from chickens in the state of Texas in the United States. However, clinical manifestations in the affected flock were consistent with avian influenza caused by a low-pathogenicity AIV and the representative virus (A/chicken/Texas/298313/04 [TX/04]) was not virulent for experimentally inoculated chickens. The hemagglutinin (HA) gene of the TX/04 isolate was similar in sequence to A/chicken/Texas/167280-4/02 (TX/02), a low-pathogenicity AIV isolate recovered from chickens in Texas in 2002. However, the TX/04 isolate had one additional basic amino acid at the HA cleavage site, which could be attributed to a single point mutation. The TX/04 isolate was similar in sequence to TX/02 isolate in several internal genes (NP, M, and NS), but some genes (PA, PB1, and PB2) had sequence of a clearly different origin. The TX/04 isolate also had a stalk deletion in the NA gene, characteristic of a chicken-adapted AIV. By analyzing viruses constructed by in vitro mutagenesis followed by reverse genetics, we found that the pathogenicity of the TX/04 virus could be increased in vitro and in vivo by the insertion of an additional basic amino acid at the HA cleavage site and not by the loss of a glycosylation site near the cleavage site. Our study provides the genetic and biologic characteristics of the TX/04 isolate, which highlight the complexity of the polygenic nature of the virulence of influenza viruses.     

Vacuolar reticulum in oomycete hyphal tips: An additional component of the Ca2+Regulatory system?

Cultures of Achlya sp., Phytophthora cinnamomi, Saprolegnia diclina, S. ferax, and S. parasitica, treated with 6-carboxyfluorescein diacetate solution, accumulate 6-carboxyfluorescein in a reticulate system of fine tubules. The network shows longitudinal polarity within the hyphae, tubules being finest toward the hyphal tips. In more mature subapical regions the network is connected with large vacuoles that also accumulate 6-carboxyfluorescein. A morphologically similar system has also been identified in freeze-substituted hyphae of S. ferax. The network is considered to be vacuolar, but differs from the tubular vacuole system of true fungi in that tubules are less motile, more frequently branched, and do not alternate with clusters of spherical vacuoles. The appearance of the network resembles patterns of calcium-sensitive dye staining and it is suggested that the vacuolar reticulum in the tip region of oomycete hyphae may act as a Ca2+ sink. The tubular reticulum in oomycetes is very fragile and can be shown with 6-carboxyfluorescein in only those hyphal tips with a motility and organelle distribution characteristic of growing hyphae with normal morphology. Diverse abnormal hyphae show a range of other fluorochrome localizations. These include large irregular compartments filled with fluorochrome, and fluorescent cytoplasm with organelles and vacuoles standing out in negative contrast. These localizations in abnormal hyphae are correlated with other structural changes indicative of damage. Special care is required in experiments with oomycetes to avoid such artefacts of localization. Copyright 1997 Academic Press. Copyright 1997 Academic Press