Anaerobic degradation of phenol via carboxylation to 4-hydroxybenzoate: in vitro study of isotope exchange between 14CO2 and 4-hydroxybenzoate

Extracts of denitrifying bacteria grown anaerobically with phenol and nitrate catalyzed an isotope exchange between ¹⁴CO₂ and the carboxyl group of 4-hydroxybenzoate. This exchange reaction is ascribed to a novel enzyme, phenol carboxylase, initiating the anaerobic degradation of phenol by para-carboxylation to 4-hydroxybenzoate. Some properties of this enzyme were determined by studying the isotope exchange reaction. Phenol carboxylase was rapidly inactivated by oxygen; strictly anoxic conditions were essential for preserving enzyme activity. The exchange reaction specifically was catalyzed with 4-hydroxybenzoate but not with other aromatic acids. Only the carboxyl group was exchanged; [U-¹⁴C]phenol was not exchanged with the aromatic ring of 4-hydroxybenzoate. Exchange activity depended on Mn²⁺ and inorganic phosphate and was not inhibited by avidin. Ortho-phosphate could not be substituted by organic phosphates nor by inorganic anions; arsenate had no effect. The pH optimum was between pH 6.5–7.0. The specific activity was 100 nmol ¹⁴CO₂ exchange · min^-1 · mg^-1 protein. Phenol grown cells contained 4-hydroxybenzoyl CoA synthetase activity (40 nmol · min^-1 · mg^-1 protein). The possible role of phenol carboxylase and 4-hydroxybenzoyl CoA synthetase in anaerobic phenol metabolism is discussed.     

Pollination biology of two species ofKielmeyera (Guttiferae) from Brazilian cerrado vegetation

The pollination biology and breeding systems ofKielmeyera coriacea andK. speciosa, two sympatric woody species common in the cerrado vegetation of C. Brazil, were studied. Both species have similar nectarless, polystemonous Papaver-type flowers which are visited by a similar spectrum of insects, though they bloom in different seasons and are thus phenologically isolated. Large carpenter bees seem to be the most important pollinators and these and other bees effect buzz pollen retrieval despite the fact that anthers are not poricidal. Both species ofKielmeyera possess strong xenogamous breeding systems. The presence of staminate flowers and andromonoecy inK. coriacea, as well as the longevity ofK. speciosa flowers are discussed as alternative strategies to improve pollination success and reproductive efficacy.     

α-[3H]Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid Binding to Rat Striatal Membranes: Effects of Selective Brain Lesions

The binding of alpha-[3H]amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ([3H]AMPA), a structural Glu analog, to rat striatal membranes was studied. In the absence of potassium thiocyanate and Cl-/Ca2+, saturation-curve analysis of [3H]AMPA binding suggested that a single class of noninteracting binding sites with a KD value of 340 +/- 27 nM was involved, although AMPA inhibition of [3H]AMPA binding set at a concentration of 100 nM suggested, in contrast, the presence of multiple populations of striatal binding sites. Several other excitatory amino acid receptor agonists and antagonists were tested, and the most potent and selective quisqualic acid (QA) receptor agonists (QA, L-Glu, and AMPA) were found to represent the most potent inhibitors of [3H]AMPA binding. N-Methyl-D-aspartate receptor agonists and antagonists were ineffective as displacers of the [3H]AMPA binding. Lesions of intrastriatal neurons (using kainic acid local injections) and of corticostriatal afferent fibers led 2-3 weeks later to large decreases (63 and 30%, respectively) in striatal [3H]AMPA binding, whereas selective lesion of the nigrostriatal dopaminergic pathway (using nigral injection of 6-hydroxy-dopamine) was without any influence. Taken together, these results suggest that [3H]AMPA binding is primarily associated with postsynaptic intrastriatal neurons. Some [3H]AMPA binding sites may also be located presynaptically on corticostriatal nerve endings. So, in addition to the possibility that [3H]AMPA binding sites may be involved in corticostriatal synaptic transmission, it is interesting that these putative QA-preferring excitatory amino acid receptor sites may also play some role in autoregulatory processes underlying this excitatory synaptic transmission.     

Expression sites and developmental regulation of genes encoding (1→3,1→4)-β-glucanases in germinated barley

Expression sites of genes encoding (13,14)--glucan 4-glucanohydrolase (EC 3.2.1.73) have been mapped in germinated barley grains (Hordeum vulgare L.) by hybridization histochemistry. A³²P-labelled cDNA (copy DNA) probe was hybridized to cryosections of intact barley grains to localize complementary mRNAs. No mRNA encoding (13,14)--glucanase is detected in ungerminated grain. Expression of (13,14)--glucanase genes is first detected in the scutellum after 1 d and is confined to the epithelial layer. At this stage, no expression is apparent in the aleurone. After 2 d, levels of (13,14)--glucanase mRNA decrease in the scutellar epithelium but increase in the aleurone. In the aleurone layer, induction of (13,14)--glucanase gene expression, as measured by mRNA accumulation, progresses from the proximal to distal end of the grain as a front moving away from, and parallel to, the face of the scutellum.Abbreviations cDNA copy DNA - RNase ribonuclease     

Glycine decarboxylase is confined to the bundle-sheath cells of leaves of C3−C4 intermediate species

Immunogold labelling has been used to determine the cellular distribution of glycine decarboxylase in leaves of C₃, C₃–C₄ intermediate and C₄ species in the genera Moricandia, Panicum, Flaveria and Mollugo. In the C₃ species Moricandia foleyi and Panicum laxum, glycine decarboxylase was present in the mitochondria of both mesophyll and bundle-sheath cells. However, in all the C₃–C₄ intermediate (M. arvensis var. garamatum, M. nitens, M. sinaica, M. spinosa, M. suffruticosa, P. milioides, Flaveria floridana, F. linearis, Mollugo verticillata) and C₄ (P. prionitis, F. trinervia) species studied glycine decarboxylase was present in the mitochondria of only the bundle-sheath cells. The bundle-sheath cells of all the C₃–C₄ intermediate species have on their centripetal faces numerous mitochondria which are larger in profile area than those in mesophyll cells and are in close association with chloroplasts and peroxisomes. Confinement of glycine decarboxylase to the bundle-sheath cells is likely to improve the potential for recapture of photorespired CO₂ via the Calvin cycle and could account for the low rate of photorespiration in all C₃–C₄ intermediate species.Abbreviation and symbol kDa kilodaltons - CO₂ compensation point     

Suberized bundle sheaths in grasses (Poaceae) of different photosynthetic types. II. Apoplastic permeability

Summary The relative hydraulic conductivities of major and minor longitudinal veins, and the apoplastic permeability of the bundle sheaths surrounding all longitudinal and transverse veins were investigated in representatives of the C₃, C₄/NAD-ME, C₄/NAD-ME/PCK intermediate, C₄/PCK and C₄/NADP-ME photosynthetic types. Using the Hagen-Poiseuille equation and measurements of tracheary element diameters, the number of elements in each vein type and the numbers of each vein type, we calculated that 87–99% of the water flow in a longitudinal direction would be expected to occur in the major veins. The permeability of the mestome sheaths and parenchymatous bundle sheaths surrounding the veins was tested using the negatively-charged, fluorescent dye, trisodium 3-hydroxy-5,8,10-pyrenetrisulfonate (PTS). This dye proved nontoxic to plant tissue at a concentration of 0.5%, according to a deplasmolysis test with onion epidermal strips. The PTS concentration achieved in the tested grass leaves was about 0.035%, well below the toxic limit. When a solution of PTS was fed to the leaves by means of a basal cut, the dye moved into the veins of all orders. From there, it moved outward into the surrounding tissues, indicating that the sheaths surrounding the veins of all orders in all species tested were permeable. Therefore, contrary to previous predictions based on structural observations and some tracer studies, bundle sheaths with suberized cell walls do not function as endodermal layers.     

Leaf ultrastructure of C4 species possessing different Kranz anatomical types in the cyperaceae

The leaf ultrastructure of NADP-malic enzyme type C₄ species possessing different anatomical features in the Cyperaceae was examined: types were the Rhynchosporoid type, a normal Kranz type in which mesophyll cells are adjacent to Kranz cells, and Fimbristyloid and Chlorocyperoid types, unusual Kranz types in which nonchlorophyllous mestome sheath intervenes between the two types of green cells. They show structural characteristics basically similar to the NADP-malic enzyme group of C₄ grasses, that is, centrifugally located chloroplasts with reduced grana and no increase of mitochondrial frequency in the Kranz cells. However, the Kranz cell chloroplasts of the Fimbristyloid and Chlorocyperoid types exhibit convoluted thylakoid systems and a trend of extensive development of peripheral reticulum, although those of the Rhynchosporoid type do not possess such particular membrane systems. The suberized lamella, probably a barrier for CO₂ diffusion, is present in the Kranz cell walls of the Rhynchosporoid type and in the mestome sheath cell walls of the other two types, and tightly surrounds the Kranz cells (sheaths) that are the sites of the decarboxylation of C₄ acids. These ultrastructural features are discussed in relation to C₄ photosynthetic function.     

Asymmetric Reduction of 4-Oxoisophorone Using Immobilized Thermophilic Bacteria Under Conditions of Controlled Cell Growth

Asymmetric reduction of 2,6,6,-trimethyl-2-cyclohexene-l,4-dione (4-oxoisophrone) to (6R)-2,2,6-trimethyl-1,4-cyclohexane-dione((3R)-dihydro-4-oxoisophorone) was catalysed by immobilized thermophilic bacteria, Thermomonospora curvata JTS 321. Because of leakage of entrapped cells from gel beads during reactions using culture medium, we optimized the medium to allow the microbial conversion under conditions of controlled cell growth. Of the media screened, liver infusion medium was found to be the most suitable and microbial conversion was achieved without cell leakage from the immobilized gels. Immobilized T. curvata cells were repeatedly used for the asymmetric reduction of 4-oxoisophorone, more than 15 times, with an extent of conversion of 50%.     

Purification of the Lewis blood-group gene associated α-3/4-fucosyltransferase from human milk: an enzyme transferring fucose primarily to Type 1 and lactose-based oligosaccharide chains

A soluble Lewis blood-group gene associated -3/4-L-fucosyltransferase has been purified from human milk by a series of steps involving hydrophobic chromatography on Phenyl Sepharose 4B, ion exchange chromatography on CM-Sephadex C-50, affinity chromatography on GDP-hexanolamine Sepharose 4B and gel filtration on Sephacryl S-200. The first step separated -3-L-fucosyltransferase activity directed towardsN-acetylglucosamine in Type 2 (Gal1-4GlcNAc-R) acceptors from an -3/4-fucosyltransferase fraction acting on both Type 1 (Gal1-3GlcNAc-R) and Type 2 acceptors. Further purification of this latter fraction on CM-Sephadex and GDP-hexanolamine Sepharose gave a single peak of fucosyltransferase activity that catalysed the addition of fucose toN-acetylglucosamine in both Type 1 and Type 2 acceptors and to theO-3 position of glucose in lactose-based oligosaccharides. The enzyme preparation at this stage resembled previously described -3/4-fucosyltransferase preparations purified from human milk. However, gel filtration of this preparation on Sephacryl S-200 or Sephadex G-150 separated further amounts of -3-fucosyltransferase activity acting solely on Type 2 acceptors and left a residual -3/4-fucosyltransferase that retained strong -4 activity with the Type 1 acceptor, lacto-N-biose 1, and -3 activity with 2-fucosyllactose, but had relatively little -3 activity withN-acetyllactosamine and virtually no capacity to transfer fucose to glycoproteins withN-linked oligosaccharide chains having unsubstituted terminal Type 2 structures.