Crystal structure of a novel mid-gut procarboxypeptidase from the cotton pest Helicoverpa armigera.

The cotton bollworm Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) is one of the most serious insect pests in Australia, India and China. The larva causes substantial economical losses to legume, fibre, cereal oilseed and vegetable crops. This pest has proven to be difficult to control by conventional means, mainly due to the development of pesticide resistance. We present here the 2.5 A crystal structure from the novel procarboxypeptidase (PCPAHa) found in the gut extracts from H. armigera larvae, the first one reported for an insect. This metalloprotease is synthesized as a zymogen of 46.6 kDa which, upon in vitro activation with Lys-C endoproteinase, yields a pro-segment of 91 residues and an active carboxypeptidase moiety of 318 residues. Both regions show a three-dimensional structure quite similar to the corresponding structures in mammalian digestive carboxypeptidases, the most relevant structural differences being located in the loops between conserved secondary structure elements, including the primary activation site. This activation site contains the motif (Ala)(5)Lys at the C terminus of the helix connecting the pro- and the carboxypeptidase domains. A remarkable feature of PCPAHa is the occurrence of the same (Ala)(6)Lys near the C terminus of the active enzyme. The presence of Ser255 in PCPAHa instead of Ile and Asp found in the pancreatic A and B forms, respectively, enlarges the S1' specificity pocket and influences the substrate preferences of the enzyme. The C-terminal tail of the leech carboxypeptidase inhibitor has been modelled into the PCPAHa active site to explore the substrate preferences and the enzymatic mechanism of this enzyme.     

Seminiferous tubule transfection in vitro to define post-meiotic gene regulation

Background  

Post-meiotically expressed genes in the testis are essential for the proper progression of spermatogenesis, and yet, aside from the construction of individual transgenic mice using specific promoters to drive reporter plasmids, there are only very limited possibilities for relevant and quantitative analysis of gene promoters. This is due to the special nature of post-meiotic haploid cells, which to date are not represented in any appropriate cell-lines. This article reports the development of novel methodology using isolated and cultured rat seminiferous tubules in a multiwell format, into which promoter-reporter constructs can be introduced by a combination of microinjection and electroporation.     

Purification and isolation of the arom aggregates of Schizosaccharomyces pombe

The five enzymes that catalyzing steps two through six in the prechorismate polyaromatic amino acid biosynthetic pathway are physically associated and have been purified up to 400-fold from Schizosaccharomyces pombe. The native arom aggregate has a molecular weight of approx. 140,000-145,000 based on gel filtration, glycerol-density-gradient centrifugation, and polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. Similarities between the S. pombe arom aggregate and that of Neurospora crassa and Euglena gracilis are discussed.     

Inter- and intramolecular crosslinks in histone H3 induced by 5,5'-dithiobis(2-nitrobenzoic acid).

The primary reaction between the cysteine residues of histone H3 and dithiobis-(nitrobenzoic acid) may be succeeded by thiol-disulfide interchange steps leading to intraor intermolecularly crosslinked H3 molecules. A chromatographic assay is applied to detect consecutive reactions of this type and is used to derive conditions by which they are promoted or suppressed. Elimination of the secondary interchange is shown to be essential if the kinetics of the reaction is to be interpreted.     

Ciliary receptors associated with the mouth and pharynx of Acoela (Acoelomorpha): a comparative ultrastructural study

The ultrastructure and distribution of receptor cells near the mouth and (where present) the pharynx of Hofstenia miamia, Proporus bermudensis, Conaperta thela, and Convoluta convoluta (Acoela) were investigated by transmission electron microscopy and confocal laser scanning microscopy of specimens stained with a fluorescence marker for actin. Five types of monociliary receptors were identified: (1) non‐collared receptors with a single long and narrow ciliary rootlet; (2) non‐collared receptors with a wide main ciliary rootlet and a smaller posterior rootlet; (3) non‐collared receptors with a single wide and hollow ciliary rootlet with a granulated core; (4) Collar (?) receptors with obliquely radial filament bundles in the cell apex and with a single hollow ciliary rootlet composed of numerous strand‐like elements; and (5) Collar receptors lacking a striated rootlet but with a granular body (swallow's nest rootlet). While H. miamia bears the first two receptor types, P. bermudensis has receptors of type 1, 3 and 5, and Cona. thela and Conv. convoluta have receptors of type 3, 4 and 5. The density of receptors is generally highest at the anterior body tip, regardless of where the mouth is located. Most receptor types occur scattered over the whole body but type 2 receptors of H. miamia are restricted to the pharynx and mouth region. The lack of a common receptor type specific for the mouth and pharynx of the investigated species points to an independent origin of the pharynges in Hofsteniidae and in Proporidae and of the mouth tube in Convolutidae. Moreover, the homology of the so‐called collar receptors in Acoela with typical collar receptors in other invertebrates is questioned.     

Refined three-dimensional structures of two cyanobacterial C-phycocyanins at 2.1 and 2.5 A resolution. A common principle of phycobilin-protein interaction

The crystal structure of the light-harvesting protein-pigment complex C-phycocyanin (C-PC) from Mastigocladus laminosus (at 2.1 A resolution (1 A = 0.1 nm] has been refined by energy-restrained least-squares methods to a conventional R-factor of 21.7%. In the same way, the crystal structure of C-PC from Agmenellum quadruplicatum has been refined further (2.5 A, R = 18.4%); pyrrole rings C and D of the chromophore at position A84 have been corrected with respect to the previously reported structure. The two C-PC structures are very similar, 213 C alpha positions have a root-mean-square deviation of 0.49 A. Polar and ionic side-chain interactions are discussed in detail and the two subunits of C-PC from M. laminosus are compared to each other. All three chromophores are completely defined and their tetrapyrroles exhibit very similar geometry. The structure of a C-PC chromophore resembles a cleaved porphyrin which has been twisted roughly 180 degrees around the C-5-C-6 and C-14-C-15 bonds. Accordingly, the configuration/conformation of the chromophores is Z-anti, Z-syn, Z-anti (with the exception of the "configuration" of C-15 of chromophore B155, which is almost midway between Z and E). The three chromophores interact similarly with the protein. They arch around aspartate residues (A87, B87 and B39), and the nitrogens of pyrroles B and C are within hydrogen-bonding distance of one of the carboxylate oxygens. Most of the propionic side-chains of the chromophores form salt bridges with arginine and lysine residues. The updated relative chromophore distances and orientations confirm our conclusion that hexameric aggregates are probably the basic functional units, and that inter-hexameric energy transfer takes place preferentially via the central B84 chromophores.     

On the origin of the limited control of mitochondrial respiration by the adenine nucleotide translocator

A thermodynamic control theory previously developed has been applied to mitochondrial oxidative phosphorylation with emphasis on the role of delta microH and coupling and within the paradigm of delocalized chemiosmotic coupling. The basis for the observed distribution of flux control over the participating enzymes is shown to lie in the relative magnitudes of so-called delta microH elasticity coefficients, i.e., the delta microH dependencies of the different mitochondrial processes. In particular the relatively strong delta microH dependence of mitochondrial respiration is responsible for the significant role of the adenine nucleotide translocator in the control of oxidative phosphorylation. Uncoupling decreases the control exerted by this translocator on respiration but increases that exerted on phosphorylation.     

The high-resolution X-ray crystal structure of the complex formed between subtilisin Carlsberg and eglin c, an elastase inhibitor from the leech Hirudo medicinalis. Structural analysis, subtilisin structure and interface geometry

Triclinic crystals of the complex formed by eglin with subtilisin Carlsberg were analyzed by X-ray diffraction. The crystal and molecular structure of this complex was determined with data that extended to 0.12-nm resolution by a combination of Patterson search methods and isomorphous replacement techniques. Its structure was refined to a crystallographic R value of 0.178 (1.0-0.12 nm) using an energy-restraint least-squares procedure. The complete subtilisin molecule could be traced without ambiguity in the refined electron density. The eglin component, from which an amino-terminal segment is cleaved off, is only defined from Lys8I (i.e. the lysine residue 8 of the inhibitor) onwards. Per unit cell, 436 fixed solvent molecules and 2 calcium ions were located. In spite of 84 amino acid replacements and one deletion, subtilisin Carlsberg exhibits a very similar polypeptide fold to subtilisin BPN'. The root-mean-square deviations of all alpha-carbon atoms (excluding those at the deletion site) from models of subtilisin BPN' [Alden, R. A., Birktoft, J. J., Kraut, J., Robertus, J. D. & Wright, C. S. (1971) Biochem. Biophys. Res. Commun. 45, 337-344] and subtilisin Novo [Drenth, J., Hol, W. G. J., Jansonius, J. N. & Kockoek, R. (1972) Eur. J. Biochem. 25, 177-181] are 0.077 nm and 0.103 nm. Most of these deviations result from global shifts rather than changes of the local geometry. The single-residue deletion at position 56 affects only the surrounding conformation. Two sites of high electron density and close distances to surrounding oxygen ligands have been found in the Carlsberg enzyme which are probably occupied by calcium ions. Eglin consists of a twisted four-stranded beta-sheet flanked by an alpha-helix and by an exposed proteinase binding loop on opposite sides. Around the reactive site, Leu45I-Asp46I, this loop is mainly stabilized by electrostatic/hydrogen bond interactions with the side chains of two arginine residues which project from the hydrophobic core [Bode, W., Papamokos, E., Musil, D., Seemüller, W. & Fritz, H. (1986) EMBO J. 5, 813-818]. The reactive site loop conformation resembles that found in other 'small' proteinase inhibitors. The scissile peptide bond is not cleaved but its carbonyl group is slightly distorted from planar geometry. Most of the intermolecular contacts are contributed by the nine residues of the reactive-site loop Gly40I-Arg48I.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inducible nitrate reductase of rice plants as a possible indicator for nitrification in water-logged paddy soils

When following the pattern of the disappearance of NH ₄ ⁺ –N from ammonium sulfate applied to the flooded soil-rice plant system (field and greenhouse experiments) during a growing season, it was observed that the lowest NH ₄ ⁺ –N level coincided with the highest value of NR activity in the leaves. Nitrate was detected in both the root and shoot systems of the rice plants and autotrophic nitrifiers (Nitrosomonas and Nitrobacter) were particularly abundant. Since it was also demonstrated in this work that the NR activity of rice plants grown with nitrate fertilization (growth chamber culture experiments) was inducible by its substrate, it can be assumed that NH ₄ ⁺ –N oxidation takes place in the water-logged soil studied. Therefore, the occurrence of the nitrification process following NH ₄ ⁺ –N fertilizer application can be predicted by thein vitro orin situ evaluation of the NR activity of the rice leaf as an indicator.     

Seasonal and inter-annual variations of nitrogen diagenesis in the sediments of a recently impounded basin

The Méry-sur-Oise (France) storage reservoir is an artificial basin of 9 m average depth, fed by water from the river Oise with a mean residence time of about 4 days. Sediments are accumulating at a rate of about 0.7 cm/month. In the sediments, two fractions of organic nitrogen with different rates of bacterial degradation could be distinguished, one associated with fresh phytoplankton, the other made of detrital and more refractory compounds. The fluxes of oxygen, nitrate and ammonium across the sediment-water interface were measured with a bell-jar system at different seasons during a 3 year period following flooding of the basin. The measurements show clear seasonal variations in relation with the variations of temperature and input of fresh phytoplanktonic material to the sediment. In addition, a long term trend of increasing ammonium was observed. Measurements were also carried out after dredging of all accumulated sediments of the basin. They showed a considerable reduction of the flux of nitrate to the sediments and a significant reduction of the flux of ammonium to the water column.These results are interpreted in the light of a non stationary model of N diagenesis in accumulating sediments. This model is able to predict at least the general trends of benthic N cycling of basins during the early stage of their ecological succession.