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.     

Partial trisomy 20p resulting from a recombination of a familial pericentric inversion

Summary Recombination of an inherited pericentric inversion of chromosome 20 has given rise to a child with partial trisomy 20p. To our knowledge no previous familial inversions of this chromosome have been described in the literature.     

Inhibition of yeast respiration and fermentation by benomyl, carbendazim, isocyanates, and other fungicidal chemicals

The inhibition of yeast (Saccharomyces cerevesiae) metabolism by fungicidal chemicals was investigated. Glucose- or ethanol-dependent yeast respiration was measured with an oxygen electrode, and manometric determination of carbon dioxide release was used to measure fermentation. Both respiration and fermentation were inhibited more by benomyl than by identical molar concentrations of its breakdown product, carbendazim. Butyl isocyanate, another benomyl breakdown product, inhibited respiration more but inhibited fermentation less than the parent compound. Of the isocyanates tested, hexyl isocyanate was the most inhibitory towards both activities. Captan was more active and iprodione less active than benomyl. Because benomyl rapidly broke down to carbendazim when it was prepared in 80% ethanol, only 59% of the dissolved benomyl was intact when it was added to yeast to determine its effect on respiration or fermentation.     

l-Glutamate-Dependent Medium Alkalinization by Asparagus Mesophyll Cells : Cotransport or Metabolism?

Mechanically isolated Asparagus sprengeri Regel mesophyll cells cause alkalinization of the suspension medium on the addition of l-glutamate or its analog l-methionine-d,l-sulfoximine. Using a radiolabeled pH probe, it was found that both compounds caused internal acidification whereas l-aspartate did not. Fusicoccin stimulated H⁺ efflux from the cells by 111% and the uptake of l-[U-¹⁴C]glutamate by 55%. Manometric experiments demonstrated that, unlike l-methionine-d,l-sulfoximine, l-glutamate stimulated CO₂ evolution from nonilluminated cells. Simultaneous measurements of medium alkalinization and ¹⁴CO₂ evolution upon the addition of labeled l-glutamate showed that alkalinization was immediate and reached a maximum value after 45 minutes whereas ¹⁴CO₂ evolution exhibited a lag before its appearance and continued in a linear manner for at least 100 minutes. Rates of alkalinization and uptake of l-[U-¹⁴C]glutamate were higher in the light while rates of ¹⁴CO₂ evolution were higher in the dark. The major labeled product of glutamate decarboxylation, γ-aminobutyric acid, was found in the cells and the suspension medium. Its addition to the cell suspension did not result in medium alkalinization and evidence indicates that it is lost from the cell to the medium. The data suggest that the origin of medium alkalinization is co-transport not metabolism, and that the loss of labeled CO₂ and γ-aminobutyric acid from the cell result in an overestimation of the stoichiometry of the H⁺/l-glutamate uptake process.     

Biosynthesis of riboflavin. Enzymatic formation of 6,7-dimethyl-8-ribityllumazine from pentose phosphates

The xylene ring of riboflavin originates by dismutation of the precursor, 6,7-dimethyl-8-ribityllumazine. The formation of the latter compound requires a 4-carbon unit as the precursor of carbon atoms 6 alpha, 6, 7, and 7 alpha of the pyrazine ring. The formation of riboflavin from GTP and ribose phosphate by cell extract from Candida guilliermondii has been observed by Logvinenko et al. (Logvinenko, E. M., Shavlovsky, G. M., Zakal'sky, A. E., and Zakhodylo, I. V. (1982) Biokhimiya 47, 931-936). We have studied this enzyme reaction in closer detail using carbohydrate phosphates as substrates and synthetic 5-amino-6-ribitylamino-2,4-(1H,3H)-pyrimidinedione or its 5'-phosphate as cosubstrates. Several pentose phosphates and pentulose phosphates can serve as substrate for the formation of riboflavin with similar efficiency. The reaction requires Mg2+. Various samples of ribulose phosphate labeled with 14C or 13C have been prepared and used as enzyme substrates. Radioactivity was efficiently incorporated into riboflavin from [1-14C]ribulose phosphate, [3,5-14C]ribulose phosphate, and [5-14C]ribulose phosphate, but not from [4-14C]ribulose phosphate. Label from [1-13C]ribose 5-phosphate was incorporated into C6 and C8 alpha of riboflavin. [2,3,5-13C]Ribose 5-phosphate yielded riboflavin containing two contiguously labeled segments of three carbon atoms, namely 5a, 9a, 9 and 8, 7, 7 alpha. 5-Amino-6-[1'-14C] ribitylamino-2,4 (1H,3H)-pyrimidinedione transferred radioactivity exclusively to the ribityl side chain of riboflavin in the enzymatic reaction. It follows that the 4-carbon unit used for the biosynthesis of 6,7-dimethyl-8-ribityllumazine consists of the pentose carbon atoms 1, 2, 3, and 5 in agreement with earlier in vivo studies.     

Biosynthesis of riboflavin in Bacillus subtilis: origin of the four-carbon moiety.

We studied the incorporation of [1-13C]ribose and [1,3-13C2]glycerol into the riboflavin precursor 6,7-dimethyl-8-ribityllumazine, using a riboflavin-deficient mutant of Bacillus subtilis. The formation of the pyrazine ring requires the addition of a four-carbon moiety to a pyrimidine precursor. The results show that C-6 alpha, C-6, C-7, and C-7 alpha of 6,7-dimethyl-8-ribityllumazine were biosynthetically equivalent to C-1, C-2, C-3, and C-5 of a pentose phosphate. C-4 of the pentose precursor was lost through an intramolecular skeletal rearrangement. Thus, the last steps in the biosynthesis of 6,7-dimethyl-8-ribityllumazine apparently involve the same mechanism in bacteria as in fungi.     

Sequence of a novel plant ras-related cDNA from Pisum sativum

A clone isolated from a purple podded pea (Pisum sativum L.) cDNA library was shown to contain the complete coding sequence of a polypeptide with considerable homology to various members of the ras superfamily. The ras superfamily are a group of monomeric GTP-binding proteins of 21–25 kDa found in eukaryotic cells. Conserved sequences in the isolated clone include the GTP-binding site, GDP/GTP hydrolysis domain and C-terminal Cys residues involved in membrane attachment. Comparisons of the predicted amino acid sequence with those of other ras proteins show significantly higher homologies (ca. 70%) to two mammalian gene products, those of the BRL-ras oncogene, and the canine rab7 gene, than to any of the plant ras gene products so far identified (<40% homology). The high percentage of amino acid identity suggests that this cDNA may be the product of a gene, designated Psa-rab, which is the plant counterpart of rab7. Rab/ypt proteins are a subfamily of the ras superfamily thought to be involved in intracellular transport from the endoplasmic reticulum to the Golgi apparatus and in vesicular transport.Northern blot hybridisation analysis of total RNA from green and purple podded pea revealed a mRNA species of approximately the same size as the isolated cDNAs.     

Billingen (Lower Arenig/Lower Ordovician) acritarchs from the East European Platform and their palaeobiogeographic significance

Billingen (Lower Arenig/Lower Ordovician) sediments of the St. Petersburg region, northwest Russia and the Leba area, northern Poland of the East European Craton yield acritarch assemblages, which are largely homogenous though displaying minor compositional differences that probably reflect a gradient from inner to outer shelf environments. Comparison with coeval acritarch microflora from the Yangtze Platform, South China, shows an overall similarity between Baltoscandian and South Chinese phytoplankton. The widespread uniformity in the fossil microphytoplankton may be related to the extensive global 'evae' sea-level transgression, which characterized the Billingen time. This suggests that during the Tremadoc through early Arenig times, acritarch assemblages displayed essentially an undifferentiated cold-water and oceanic character along the whole margin of Perigondwana in the South, as well as on the South Chinese and Baltic platforms, at middle latitudes (Mediterranean oceanic Realm). Despite this overall similarity, however, some typical taxa of the high-latitude Mediterranean Province (Arbusculidium, Coryphidium and Striatotheca) occur in South China, but are absent in Baltica. This discrepancy is explained as caused by differences in climatic and physiographic conditions that prevailed at the two palaeocontinents at this time. The inferred pattern of oceanic circulation during the Lower Ordovician is consistent with the palynological evidence of a prevailing warmer climate in Baltica than in South China, although the two palaeocontinents occupied the same palaeolatitudinal position.     

An investigation into the roles of photosynthesis and respiration in h efflux from aerated suspensions of asparagus mesophyll cells

Aerated and stirred suspensions of mechanically isolated Asparagus sprengeri Regel mesophyll cells were used to investigate the roles of respiration and photosynthesis in net H⁺ efflux. Rates varied between 0.12 and 1.99 nanomoles H⁺ per 10⁶ cells per minute or 3 and 40 nanomoles H⁺ per milligram chlorophyll per minute. The mean rate of H⁺ efflux was 10% greater in the dark. 3-(3,4-Dichlorophenyl)-l,l-dimethylurea, an inhibitor of noncyclic photophosphorylation, did not inhibit H⁺ efflux from illuminated cells. Bubbling with N₂ or addition of oligomycin, an inhibitor of mitochondrial ATP production, resulted in rapid and virtually complete inhibition of H⁺ efflux in light or dark. In the absence of aeration, H⁺ efflux came to a halt but resumed with aeration or illumination. When aeration was switched to CO₂-free air, rates of H⁺ efflux were reduced 43% in the dark and 57% in the light. Oligomycin eliminated dark CO₂ fixation but not photosynthetic CO₂ fixation. It is suggested that H⁺ efflux is dependent on respiration and dark CO₂ fixation, but independent of photosynthesis.