Production of hydroxy fatty acids in the seeds of Arabidopsis thaliana

Seed-specific expression in Arabidopsis thaliana of oleate hydroxylase enzymes from castor bean and Lesquerella fendleri resulted in the accumulation of hydroxy fatty acids in the seed oil. By using various Arabidopsis mutant lines it was shown that the endoplasmic reticulum (ER) n-3 desaturase (FAD3) and the FAE1 condensing enzyme are involved in the synthesis of polyunsaturated and very-long-chain hydroxy fatty acids, respectively. In Arabidopsis plants with an active ER Delta12-oleate desaturase the presence of hydroxy fatty acids corresponded to an increase in the levels of 18:1 and a decrease in 18:2 levels. Expression in yeast indicates that the castor hydroxylase also has a low level of desaturase activity.     

Salt-tolerant phenol-degrading microorganisms isolated from Amazonian soil samples

Two phenol-degrading microorganisms were isolated from Amazonian rain forest soil samples after enrichment in the presence of phenol and a high salt concentration. The yeast Candida tropicalis and the bacterium Alcaligenes faecoalis were identified using several techniques, including staining, morphological observation and biochemical tests, fatty acid profiles and 16S/18S rRNA sequencing. Both isolates, A. faecalis and C. tropicalis, were used in phenol degradation assays, with Rhodococcus erythropolis as a reference phenol-degrading bacterium, and compared to microbial populations from wastewater samples collected from phenol-contaminated environments. C. tropicalis tolerated higher concentrations of phenol and salt (16 mM and 15%, respectively) than A. faecalis (12 mM and 5.6%). The yeast also tolerated a wider pH range (3-9) during phenol degradation than A. faecalis (pH 7-9). Phenol degradation was repressed in C. tropicalis by acetate and glucose, but not by lactate. Glucose and acetate had little effect, while lactate stimulated phenol degradation in A. faecalis. To our knowledge, these soils had never been contaminated with man-made phenolic compounds and this is the first report of phenol-degrading microorganisms from Amazonian forest soil samples. The results support the idea that natural uncontaminated environments contain sufficient genetic diversity to make them valid choices for the isolation of microorganisms useful in bioremediation.     

Human pyridoxal kinase: overexpression and properties of the recombinant enzyme

Pyridoxal kinase catalyses the phosphorylation of the vitamin B6. A human brain pyridoxal kinase cDNA was isolated, and the recombinant enzyme was overexpressed in E. coli as a fusion protein with maltose binding protein (MBP). Pure pyridoxal kinase exhibits a molecular mass of about 40 kDa when examined by SDS-PAGE and FPLC gel filtration. The recombinant enzyme is a monomer endowed with catalytic activity, indicating that the native quaternary structure of pyridoxal kinase is not a prerequisite for catalytic function. Zn2+ is the most effective divalent cation in the phosphorylation of pyridoxal, and the human enzyme has maximum catalytic activity in the narrow pH range of 5.5-6.0. The Km values for two substrates pyridoxal and ATP are 97 microM and 12 microM, respectively. In addition, the unfolding processes of the recombinant enzyme were monitored by circular dichroism. The values of the free energy change of unfolding (AGo = 1.2 kcal x mol(-1) x K(-1)) and the midpoint transition (1 M) suggested that the enzyme is more stable than ovine pyridoxal kinase against denaturation by guanidine hydrochloride. Intrinsic fluorescence spectra of the human enzyme from red-edge excitation and fluorescence quenching experiments showed that the tryptophanyl residues are not completely exposed and more accessible to neutral acrylamide than to the negatively charged iodide. The first complete set of catalytic and structural properties of human pyridoxal kinase provide valuable information for further biochemical studies on this enzyme.     

Mitochondrial membrane depolarization and the selective death of dopaminergic neurons by rotenone: protective effect of coenzyme Q10

Chronic exposure to the pesticide rotenone induces a selective degeneration of nigrostriatal dopaminergic neurons and reproduces the features of Parkinson's disease in experimental animals. This action is thought to be relevant to its inhibition of the mitochondrial complex I, but the precise mechanism of this suppression in selective neuronal death is still elusive. Here we investigate the mechanism of dopaminergic neuronal death mediated by rotenone in primary rat mesencephalic neurons. Low concentrations of rotenone (5-10 nM) induce the selective death of dopaminergic neurons without significant toxic effects on other mesencephalic cells. This cell death was coincident with apoptotic events including capsase-3 activation, DNA fragmentation, and mitochondrial membrane depolarization. Pretreatment with coenzyme Q10, the electron transporter in the mitochondrial respiratory chain, remarkably reduced apoptosis as well as the mitochondrial depolarization induced by rotenone, but other free radical scavengers such as N-acetylcysteine, glutathione, and vitamin C did not. Furthermore, the selective neurotoxicity of rotenone was mimicked by the mitochondrial protonophore carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), a cyanide analog that effectively collapses a mitochondrial membrane potential. These data suggest that mitochondrial depolarization may play a crucial role in rotenone-induced selective apoptosis in rat primary dopaminergic neurons.     

BAALC 1-6-8 protein is targeted to postsynaptic lipid rafts by its N-terminal myristoylation and palmitoylation, and interacts with alpha, but not beta, subunit of Ca/calmodulin-dependent protein kinase II

We cloned a rat BAALC 1-6-8 isoform cDNA (GenBank Accession No. AB073318) that encoded a 22-kDa protein, and identified endogenous BAALC 1-6-8 protein in the brain. The gene was expressed widely in the frontal part of the brain, and the protein was localized to the synaptic sites and was increased in parallel with synaptogenesis. The protein interacted with the alpha, but not beta, subunit of Ca(2+)/calmodulin-dependent protein kinase II (CaMKIIalpha). The interaction occurred between the N-terminal 35-amino-acid region of BAALC 1-6-8 protein and the C-terminal end of the regulatory domain of CaMKIIalpha, which contains alpha isoform-specific sequence. Thus, the interaction may be CaMKIIalpha-specific. We also found that BAALC 1-6-8 protein, as well as CaMKIIalpha, was localized to lipid rafts and that both myristoylation and palmitoylation of BAALC 1-6-8 N-terminal portion were required for targeting of the protein into lipid rafts. These findings suggest that BAALC 1-6-8 protein play a synaptic role at the postsynaptic lipid raft possibly through interaction with CaMKIIalpha.     

Reservoir and conduit function of right atrium: impact on right ventricular filling and cardiac output

The purpose of this study was to investigate the relationship between right atrial (RA) reservoir and conduit function and to determine how hemodynamic changes influence this relationship and its impact on cardiac output. In 11 open-chest sheep, RA reservoir and conduit function were quantified as RA inflow with the tricuspid valve closed versus open, respectively. Conduit function was separated into early (before A wave) and late (after A wave) components. The effects of inotropic stimulation, partial pulmonary artery occlusion, and pericardiotomy were tested. At baseline with the pericardium intact, reservoir function accounted for 0.56 (SD 0.13) of RA inflow, early conduit for 0.29 (SD 0.07), and late conduit (during RA contraction) for 0.16 (SD 0.11). Inotropic stimulation decreased conduit function and increased reservoir function, but these effects did not reach statistical significance. With partial pulmonary artery occlusion, early conduit function fell to 0.20 (SD 0.11) (P < 0.04), and the conduit-to-reservoir ratio decreased by 41% (P < 0.03). Similarly, after pericardiotomy, early conduit function fell to 0.14 (SD 0.09) (P < 0.004), reservoir function increased to 0.72 (SD 0.08) (P < 0.04), and, consequently, the early conduit-to-reservoir ratio decreased by 63% (P < 0.006). Cardiac output was inversely related to the conduit-to-reservoir ratio (r = 0.39, P < 0.001). This study demonstrated that the right atrium adjusts its ability to act more as a reservoir than a conduit in a dynamic manner. The RA conduit-to-reservoir ratio was directly related to the right ventricular pressure-RA pressure gradient at the time of maximum RA volume, with increased ventricular pressures favoring conduit function, but it was inversely related to cardiac output, with an increase in the reservoir contribution favoring improved cardiac output.     

<Emphasis Type="BoldItalic">Phellinus linteus</Emphasis> grown on germinated brown rice suppresses IgE production by the modulation of Th1/Th2 balance in murine mesenteric lymph node lymphocytes

We compared the immunomodulating effects of Phellinus linteus (PL), germinated brown rice (BR) and P.␣linteus grown on germinated brown rice (PB) on IgE production in murine mesenteric lymph node (MLN) lymphocytes. All extracts decreased IgE concentrations by 43–65% compared to control mice in both serum and MLN lymphocytes. In addition, PL and PB increased the proportion of CD4⁺ T cells by␣9% and 12% in MLN lymphocytes. IFN-γ concentration, Th1 cytokine, was significantly increased by 44–67%, whereas IL-4 and IL-10 concentrations, Th2 cytokine, significantly decreased by 30–60% in the three treated groups compared to control group. These results suggest that PB suppresses IgE production through the modulation of Th1/Th2 balance to down-regulate Th2 response in MLN lymphocytes, even though a synergistic effect of PB was not found.