Conformational Analysis of the Thrombin Receptor Agonist Peptides SFLLR and SFLLR-NH2 by NMR: Evidence for a Cyclic Bioactive Conformation

The conformational properties of the pentapeptide Ser-Phe-Leu-Leu-Arg (P5), a human thrombin receptor-derived sequence forming part of a tethered ligand which activates the thrombin receptor, and its more active amide derivative Ser-Phe-Leu-Leu-Arg-NH₂ (P5-NH₂), have been studied by proton NMR spectroscopy in dimethylsulfoxide. Measurements of nuclear Overhauser effects, performed using two-dimensional rotating frame nuclear Overhauser (ROESY) and one-dimensional nuclear Overhauser enhancement (NOE) spectroscopy, revealed that P5 exists mainly in an extended conformation. However, proton–proton 1D-NOEs between Phe CH and Ser CH, Leu³ CH and Leu³ NH, and Leu⁴ CH and Leu⁴ NH, as well as between the Ser and Arg sidechains, also implicated a minor conformer for P5 having a curved backbone and a near-cyclic structure. In contrast to P5, measurements of NOEs and ROEs for P5-NH₂ revealed a more stabilized cyclic structure which may account for its higher biological potency. Thus strong interresidue sequential NH (i)–NH (i + 1) interactions, as well as C-terminal carboxamide to N-terminal side-chain interactions, i.e., Arg CONH₂ to Phe ring and Arg CONH₂ to Ser , observed at lower levels of the ROESY spectrum, supported a curved backbone structure for SFLLR-NH₂. Since the higher potaency P5-NH₂ analogue adopts predominantly a cyclic structure, a cyclic bioactive conformation for thrombin receptor agonist peptides is suggested.     

Distance-constraint approach to protein folding. I. Statistical analysis of protein conformations in terms of distances between residues

A statistical analysis of protein conformations in terms of the distance between residues, represented by their C atoms, is presented. We consider four factors that contribute to the determination of the distanced _i,i+k between a given pair ofith and(i+k)th residues in the native conformation of a globular protein: (1) the distancek along the chain, (2) the size of the protein, (3) the conformational states of theith to(i+k)th residues, and (4) the amino acid types of the and(i+k)th residues. In order to account for the dependence on the distancek along the chain, the statistics are taken for three ranges, viz., short, medium, and long ranges (k8; 9k20; andk21; respectively). In the statistics of short-range distances, a mean distanceD _k and its standard deviationS _k are calculated for each value ofk, with and without taking into account the conformational states of all residues fromi toi+k (factors 1 and 3). As an Appendix, the relations for converting from the distances between residues into other conformational parameters are discussed. In the statistics of long-range distances, a reduced distanced* _ij (the actual distance divided by the radius of gyration) is used to scale the data so that they become independent of protein size, and then a mean reduced distanceD _l (a, a) and its standard deviation _l (a, a) are calculated for each amino acid pair (a, a) (factors 2 and 4). The effect of the neighboring residues along the chain on the value of the distanced* _ij is explored by a linear regression analysis between the actual reduced distanced* _ij and the mean value over theD _l for all possible pairs of residues in the two segments of the (i–2)th to the (i+2)th and the (j–2)th to the (j+2)th residues. The effect is assessed in terms of the tangentA _l (a, a) of the calculated regression line for each amino acid pair (a, a). In the statistics of medium-range distances, only factors 1 and 4 are considered, to simplify the analysis. The scaled distanced _i,i+k =(d _i,i+k -D _k )/S _k is used to eliminate the dependence onk, the distance along the chain. The propertiesD _m (a, a), _m (a, a) andA _m (a, a) corresponding toD _l (a, a), _l (a, a), andA _l (a, a), and also calculated for each amino acid pair (a, a). The results are interpreted as follows: the smaller values ofD _l (a, a) andD _m (a, a) indicate a preference of the pair (a, a) for a contact (e.g., pairs between hydrophobic amino acids, and pairs of Cys with aromatic amino acids), and the larger values of these quantities indicate a preference for distant mutual location (e.g., pairs between strong hydrophilic amino acids); the smaller values of _l (a, a) and _m (a, a) indicate a strong preference for either contact or noncontact (e.g., pairs between hydrophobic amino acids, and pairs between strong hydrophobic and hydrophilic amino acids, respectively), and the larger values of these quantities indicate the ambivalent/neutral nature of the preference for contact and noncontact (e.g., pairs containing Ser or Thr); the smaller values ofA _l (a, a) andA _m (a, a) indicate that the distance of an (a, a) pair is determined independently of the amino acid character of the neighboring residues along the chain (e.g., some pairs of Cys or Met with other amino acids) and the larger values of these quantities indicare that such amino acid character contributes strongly to the determination of the distance (e.g., pairs containing Ser or Thr, and pairs between amino acids with small side chains). The difference between the statistics for the long- and medium-range distances is also discussed; the former reflect the difference between the hydrophobic and hydrophilic character of the residues, but the latter cannot be easily interpretable only in terms of hydrophobicity and hydrophilicity. The data analyzed here are used in the optimization of an object function to compute protein conformation in a subsequent paper.     

Microbial physiology of sidechain degradation of sterols

Summary A large number of valuable starting materials for steroids synthesis (e.g. 4-androstene-3,17-dione, 1,4-androstadiene-3,17-dione, 9-hydroxy-4-androsten-17-one) have been produced by microbial transformation methods. This review helps to evaluate the microbial physiological interest of the widely used sterol sidechain degradation processes. Four inducible groups of the catabolic enzymes are involved in the sterol sidechain degradation pathway; the fatty acid -oxidation system, the -oxidase reaction, a methyl-crotonyl-CoA carboxylation system and the propionyl-CoA carboylase system.Altogether nine catabolic enzymes are involved in the -sitosterol sidechain degradation pathway. They work in 14 consecutive enzymatic steps. Summing up: three molecules of FADH₂, three molecules of propionyl-SCoA, three of NADH and one molecule of acetic acid are formed, while the sidechain of one mole of sitosterol is removed selectively. The metabolism of the propionates and the acetate yield 18 molecules of NADH and 7 molecules of FADH₂. Taking into consideration the whole process more than 80 molecules of ATP could be formed during the sitosterol sidechain degradation process.     

Isolation and partial characterization of basic proteinases from stem bromelain

Crude bromelain extracts from pineapple stems (Ananas comosus) were fractionated by two-step FPLC-cation-exchange chromatography. At least eight basic proteolytically active components were detected. The two main components F4 and F5 together with the most active proteinase fraction F9 were characterized by SDS-PAGE, mass spectroscopy, multizonal cathodal electrophoresis, partial amino acid sequence, and monosaccharide composition analysis. F9 amounts to about 2% of the total protein and has a 15 times higher specific activity against the substratel-pyroglutamyl-l-phenylanalyl-l-leucine-p-nitroanilide (PFLNA) than the main component F4. The molecular masses of F4, F5, and F9 were determined to 24,397, 24,472, and 23,427, respectively, by mass spectroscopy. Partial N-terminal amino acid sequence analysis (20 amino acids) revealed that F9 differs from the determined sequence of F4 and F5 by an exchange at position 10 (tyrosineserine) and position 20 (asparagine glycine). F4 and F5 contained fucose, N-acetylglucosamine, xylose, and mannose in ratio of 1.02.01.02.0, but only 50% of the proteins seem to be glycosylated, whereas F9 was found to be unglycosylated. Polyclonal antibodies (IgG) against F9 detected F4 and F5 with tenfold reduced reactivity. ThepH optimum of F4 and F5 was betweenpH4.0 and 4.5 and for F9 close to neutralpH. The kinetic parameters for PFLNA hydrolysis were similar for F4 (K _m 2.30 mM,k _cat 0.87 sec^–1 and F5 (K _m 2.42 mM,k _cat 0.68 sec^–1), and differed greatly from F9 (K _m 0.40 mM,k _cat 3.94 sec^–1).Dedicated to H. Tschesche, Bielefeld, Germany, on behalf of his 60th anniversary.     

Effect of<Emphasis Type="Italic"> p</Emphasis>CO<Subscript>2</Subscript> and temperature on the boron isotopic composition of the zooxanthellate coral<Emphasis Type="Italic"> Acropora</Emphasis> sp.

Culture experiments were carried out with Acropora sp. (a branching scleractinian coral) in seawater at two pCO₂ conditions (438 and 725 µatm) and two temperatures (25 and 28 °C) in order to establish the pH and temperature dependence of the boron isotopic composition of the skeleton. A clear pCO₂ effect, but no temperature effect, on the coral boron isotope composition is seen. For corals cultured at normal pCO₂ (438 µatm), the ¹¹B of the skeleton was 24.0±0.2 at 25 °C, and 23.9±0.3 at 28 °C. The values of ¹¹B measured for corals cultured at higher pCO₂ (725 µatm) were lower: 22.5±0.1, and 22.8±0.1 at 25 and 28 °C, respectively. The ¹¹B of corals cultivated at both high and normal pCO₂ conditions are consistent with a dominant pH control, and are very close to that calculated from theoretical considerations. Thus, the corals do not seem to significantly alter ambient seawater for calcification with respect to pH. Co-variation between boron and carbon isotope values is explored.Communicated by: Guest Editor A. Grottoli     

The Endosperm and the Embryo of Arabidopsis thaliana are Independently Transformed through Infiltration by Agrobacterium tumefaciens

Several experiments had indicated that in planta transformation of Arabidopsis thaliana by Agrobacterium involves the female germ line. In order to identify the precise stage at which transformation occurs we have monitored expression of a gusA reporter gene in the two products of the double fertilization of infiltrated plants. The plantlets and the remaining endosperm of seeds were separately tested after germination. It appeared that in the majority of cases only the plantlet or the endosperm were transformed. Based on transformation with two vectors borne by two different Agrobacterium strains, the minority of co-transformed plantlets and endosperm can be explained by simultaneous but independent transformation events. These results indicate that mature female gametes could be the targets of T-DNA.     

Determination of the electron self-exchange rates of blue copper proteins by super-WEFT NMR spectroscopy

An NMR approach for determining the electron self-exchange (ESE) rate constants in blue copper proteins is presented. The approach uses the paramagnetic relaxation enhancement of resonances in 1D ¹H super-WEFT spectra of partly oxidized (paramagnetic) proteins. These spectra allow a more precise determination of the relevant paramagnetic linebroadenings than conventional 1D ¹H spectra and, thus, permit a more detailed investigation of the applicability of the linebroadenings for determining the electron exchange rates. The approach was used to estimate the ESE rate constant of plastocyanin from Anabaena variabilis. It was found that, although the rate constant can be determined accurately from a series of resonances, precise but erroneous constants are obtained from the resonances of the copper-bound residues, unless a narrow splitting of these resonances caused by the presence of two conformations is taken into account. As demonstrated here, this complication can be overcome by a correct analysis of the paramagnetic broadening of the combined double signals. Because of the high resolution and specific sensitivity of the approach it should be generally applicable to estimate electron transfer rates, k, if the paramagnetic relaxation enhancement R _2p of the resonances can be determined, and the conditions kR _2p or _pkR _2p are fulfilled, _p being the frequency separation between corresponding diamagnetic and paramagnetic sites.     

Further studies on the metabolism of gibberellins (GAs) A9, A20 and A29 in immature seeds of Pisum sativum cv. progress No. 9

Seed maturation of Pisum sativum cv. Progress No. 9 proceeds more slowly in winter than in summer even when the parent plants are grown in greenhouse conditions with light-and heat-supplementation. For parent plants grown under summer and winter conditions the metabolism of [³H]GA₉ in cultured seeds is qualitatively different in seeds of equivalent age and qualitatively the same in seeds of equivalent weight. 13-Hydroxylation of [³H]GA₉[³H]GA₂₀ is restricted to early stages of seed development. 2-Hydroxylation of [³H]GA₉2-OH-[³H]GA₉ has only been observed at a stage of development after endogenous GA₉ has accumulated. 2-OH-GA₉ has been shown to be endogenous to pea and is named GA₅₁. H₂-GA₃₁ and its conjugate have not been shown to be present in pea and may be induced metabolites of [³H]GA₉. The metabolism of GA₂₀GA₂₉ is used to illustrate a technique of feeding [²H][³H]GAs in order to distinguish a metabolite from the same endogenous compound. The in vitro conversion of [³H]GA₂₀[³H]GA₂₉, and the virtual non-metabolism of [³H]GA₂₉ have been confirmed for seeds in intact fruits. These results are discussed in relation to the apparent absence of conjugated GAs in mature pea seeds.Abbreviations GA_n gibberellin A_n - GC gas chromatography - GC-MS combined gas chromatography-mass spectrometry - GC-RC combined gas chromatography-radio counting - Me methyl ester - R_T etention time - SICM selected ion current monitoring - TLC thin layer chromatography - TMS trimethyl silyl ether The author is née Frydman     

Calcium transport by pea root membranes

Calcium transport has been studied using purified endomembrane vesicles from dark-grown roots of Pisum sativum L. Membranes from a mixed microsomal (non-mitochondrial) fraction showed ATP-dependent calcium uptake which was released by the ionophore A 23187, had a pH optimum of 7.2 and required Mg²⁺ for uptake. Membranes were further purified using a rapid sucrosedensity-gradient technique yielding vesicles suitable for transport studies, and were identified using marker enzymes. Uptake by plasma membrane, tonoplast, endoplasmic reticulum and Golgi apparatus was indicated. Uptake by membranes of low density (predominantly tonoplast) had a pH optimum of 7.2–7.4 and nucleotide specificity ATP> guanosine 5-triphosphate>inosine 5-triphosphate>ADP>, while that by high-density membranes had a pH optimum of 7.5–7.9 and less specificity for ATP. The importance of regulating sucrose concentrations in calcium transport studies was demonstrated.Abbreviations ER endoplasmic reticulum - GTP guanosine 5-triphosphate - IDPase inosine diphosphatase - IIP inosine 5-triphosphate     

Purification and properties of ap-nitrobenzyl esterase fromBacillus subtilis

A procedure for purifying to homogeneity a microbially produced biocatalyst useful for deblocking intermediates in the manufacture of beta-lactam antibiotics is reported. In aqueous solution the purifiedp-nitrobenzyl (PNB) carboxy-esterase was soluble, monomeric (molecular weight: 54 000 by SDS-PAGE or by gel filtration) and exhibited an acidic pl, 4.1. The PNB carboxy-esterase catalyzed rapid ester hydrolysis for simple organic esters such as PNB-acetate, benzyl acetate and -naphthyl acetate and catalyzed deblocking (ester hydrolysis) of beta-lactam antibiotic PNB esters such as cephalexin-PNB and loracarbef-PNB. TheN-terminal amino acid sequence and the amino acid composition are reported. A serine residue is involved in ester hydrolysis: the PNB carboxy esterase was inhibited by phenylmethylsulfonyl fluoride and diethylp-nitrophenyl phosphate; one mole of diisopropyl fluorophosphate titration was required per mole of PNB carboxy-esterase for complete inhibition. When the [³H]-diisopropyl fluorophosphate-treated biocatalyst was digested with Lys C and the resulting peptides separated by HPLC, a single [³H]-labeled peptide was obtained; its amino acid sequence is reported. Inhibition of the PNB carboxy esterase by diethyl pyrocarbonate suggests that a histidinyl residue (or residues) is (are) also involved in the catalytic site of the esterase.Abbreviations used -ME -mercaptoethanol - Cf cefaclor - Cf nucleus-PNB - (6R, 7R) 7-amino-3-chloro-8-oxo-5-thia-1-azabicyclo[4.2.0]-oct-2-ene-2-carboxylic acid, (4-nitrophenyl)methyl ester - Cp cephalexin - Cp-PNB p-nitrobenzyl carboxy-ester of cephalexin - DEPC diethyl, pyrocarbonate - DFP diisopropyl fluorophosphate - DMSO dimethyl sulfoxide - DNP diethylp-nitrophenyl phosphate - EDTA ethylenediaminetetraacetic acid - EGTA ethylene, glycol-bis(aminoethyl ether) - N,N,NN tetracetic acid - Lc loracarbef - Lc-PNB p-nitrobenzyl carboxy-ester of loracarbef - Lc nucleus-PNB - (6R, 7S) 7-amino-3-chloro-8-oxo-1-azabicyclo[4.2.0]-oct-2-ene-2-carboxylic acid, (4-nitrophenyl)methyl ester - Lys C an endoproteinase specifically cleaving at C terminal lysine residues - MW_r relative molecular weight - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonylfluoride - PNB p-nitrobenzyl - PNBCE p-nitrobenzyl carboxy-esterase - SDS sodium dodecyl sulfate     

Inheritance and linkage relationships of glutamate oxaloacetate transaminase isoenzymes in apple

Summary Four zones of enzymatic activity for glutamate oxaloacetate transaminase (GOT) were found in apple tissue. A dimeric gene, GOT-1, determining the fastest migrating zone, was identified. Six alleles were found, including a near null allelle which produced detectable heterodimeric bands but not homodimeric bands. A marked deficit or absence of certain geno-types in all backcrosses and in some crosses between unrelated varieties was attributed to the close linkage (r=0.02±0.005) of GOT-1 with the incompatibility S locus. GOT-1 was also closely linked with the isocitrate dehydrogenase locus IDH-1 (0.03±0.01). Proposed incompatibility genotypes for four cultivars, and the linked GOT-1 alleles are Cox: S ₁ b/S ₂ d, Idared: S ₃ a/S ₄ c, Fiesta: S ₃ a/S ₂ d and Kent: S ₃ a/S ₁ b.The results reported in this paper are part of a PhD Thesis by the first author     

Observations on the reduction of non-activated carboxylates by Clostridium formicoaceticum with carbon monoxide or formate and the influence of various viologens

Crude extracts or supernatants of broken cells of Clostridium formicoaceticum reduce unbranched, branched, saturated and unsaturated carboxylates at the expense of carbon monoxide to the corresponding alcohols. The presence of viologens with redox potentials varying from E ₀=-295 to-650 mV decreased the rate of propionate reduction. The more the propionate reduction was diminished the more formate was formed from carbon monoxide. The lowest propionate reduction and highest formate formation was observed with methylviologen. The carbon-carbon double bond of E-2-methyl-butenoate was only hydrogenated when a viologen was present. Formate as electron donor led only in the presence of viologens to the formation of propanol from propionate. The reduction of propionate at the expense of a reduced viologen can be followed in cuvettes. With respect to propionate Michaelis Menten behavior was observed. Experiments are described which lead to the assumption that the carboxylates are reduced in a non-activated form. That would be new type of biological reduction.Non-standard abbreviations glc Gas liquid chromatography - HPLC high performance liquid chromatography - RP reverse phase; Mediators (the figures in parenthesis of the mediators are redox potentials E ₀ in mV) - CAV²⁺ carbamoylmethylviologen, 1,1-carbamoyl-4,4-dipyridinium dication (E ₀=-296 mV) - BV²⁺ benzylviologen, 1,1-dibenzyl-4,4-dipyridinium dication (E ₀=-360 mV) - MV methylviologen, 1,1-dimethyl-4,4-dipyridinium-dication (E ₀=-444 mV) - DMDQ²⁺ dimethyldiquat, 4,4-dimethyl-2,2-dipyridino-1,1-ethylendication (E ₀=-514 mV) - TMV²⁺ tetramethylviologen, 1,1,4,4-tetramethyl-4,4-dipyridinium dication (E ₀=-550 mV) - PDQ²⁺ propyldiquat, 2,2-dipyridino-1,1-propenyl dication (E ₀=-550 mV) - DMPDQ²⁺ dimethylpropyldiquat, 4,4-dimethyl-2,2-dipyridino-1,1-propenyl dication (E ₀=-656 mV) - PN productivity number=mmol product (obtained by the uptake of one pair of electrons) x (biocatalyst (dry weight) kg)^-1×h^-1     

Molecular mapping of a new gene Rrs4 CI 11549 for resistance to barley scald (Rhynchosporium secalis)

Doubled haploid (DH) progeny from a cross between the scald susceptible barley (Hordeum vulgare L.) cultivar Ingrid and the resistant accession CI 11549 (Nigrinudum) was evaluated for resistance in the pathogen Rhynchosporium secalis (Oudem) J.J. Davis. Two linked and incompletely dominant loci confer resistance CI 11549 against isolate 4004. One is an allele at the complex Rrs1 locus on chromosome 3H close to the centromere; the other is located 22 cM distally on the long arm. The latter locus is designated Rrs4. In BC₃-lines into Ingrid from CI 2222 (another Nigrinudum) resistance seems governed by one locus close to the telomeric region of chromosome 7H, probably allelic to Rrs2. In neither case did we find any trace of the recessive gene rh8 reported to be present in Nigrinudum. Various resistance donors of Ethiopian origin designated as Nigrinudum, Jet or Abyssinian were identical to a great extent with respect to markers, but differed in resistance to different isolates of scald or in barley yellow dwarf virus (BYDV) resistance. The implications for their use as differentials in scald tests and screening of germplasm collections are discussed.     

Modeling of Alternative Pathways of Electron Transport to Photosystem I in Isolated Thylakoids

Kinetics of dark decay of absorbance changes at 830 nm (₈₃₀) was examined in thylakoids isolated from leaves of pea seedlings at various concentrations of exogenous NADPH or NADH. Absorbance changes were induced by far-red light to avoid electron donation from photosystem II. In the presence of either biological reductant, the kinetics of ₈₃₀ decay reflecting dark reduction of 700⁺, the primary electron donor of photosystem I, was fitted by a single exponential term. The rate of 700⁺ reduction increased with the rise in the concentration of both NADPH and NADH. The values of K _M and V _max for 700⁺ reduction estimated from concentration dependences were 105 ± 21 M and 0.32/s for NADPH or 21 ± 8 M and 0.12/s for NADH. The rate of P700⁺ reduction by either NADPH or NADH significantly increased in the presence of rotenone, a specific inhibitor of chloroplast reductase. The value of V _max was changed only in the presence of rotenone, whereas K _m was practically unaffected. Unlike the chloroplasts of intact leaves, the only enzyme mediating the input of reducing equivalents from NADPH or NADH to the electron transport chain was concluded to be present in thylakoids.     

Glycan modification of a thermostable recombinant (1-3,1-4)-β-glucanase secreted fromSaccharomyces cerevisiae is determined by strain and culture conditions

High level biosynthesis and secretion of the thermostable hybrid (1-3,1-4)--glucanase H(A16-M) has been achieved inSaccharomyces cerevisiae by means of the yeast vacuolar endoprotease B promoter (PRB1_p) and theBacillus macerans (1-3,1-4)--glucanase signal peptide. The N-glycans present on the yeast-secreted H(A16-M), denoted H(A16-M)-Y, were released by endoglycosidase H, and identified by proton NMR spectroscopy to be a homologous series of Man_8-13GlcNAc₂, although only traces of Man₉GlcNAc₂ were found. Therefore, processing of N-glycans on H(A16-M)-Y is similar to that on homologous proteins. Most of the N-glycans (88%) were neutral while the remainder were charged due to phosphorylation. Site-directed mutagenesis of Asn to Gln in two of the N-glycosylation sequons, and subsequent analysis of the N-glycans on the yeast-secreted proteins together with analysis of the N-glycans from the individual sites of H(A16-M)-Y suggest the presence of steric hindrance to glycan modification by the glycans themselves. H(A16-M)-Y produced under control of either the yeast protease B or the yeast 3-phosphoglycerate kinase promoter, each in two differentSaccharomyces strains revealed a dependence of N-glycan profile on both strain and culture conditions. The extent of O-glycosylation was found to be nine mannose units per H(A16-M)-Y molecule. An attempt to identify the linkage-sites for the O-glycans by amino acid sequencing failed, suggesting non-stoichiometric or heterogeneous O-glycosylation. The possible modes in which N-glycans might contribute to resistance of H(A16-M)-Y to irreversible thermal denaturation are discussed with respect to structural information available for H(A16-M)-Y. Abbreviations: AMY,B. amyloliquefaciens (1-3,1-4)--glucanse; MAC,B. macerans (1-3,1-4)--glucanase H(A16-M), H(A36-M), H(A78-M),H(A107-M) and H(A152-M), hybrid (1-3,1-4)--glucanases containing 16, 36, 78, 107 and 152 N-terminal amino acids, respectively, derived from AMY with the remaining amino acids derived from MAC; similar enzyme abbreviations followed by Y, e.g. H(A16-M)-Y, denote the enzymes secreted from yeast cells; PCR, polymerase chain reaction; PGK_p, yeast 3-phosphoglycerate kinase promoter; PRB1_p, yeast protease B promoter; LB, Luria-Bertani medium; SC, minimal medium; CNBr, cyanogen bromide; Endo H_f, endoglycosidase H fusion protein; PNGase F, peptide:N-glycosidase F; HPAEC; high pH anion exchange chromatography; HVE, high voltage paper electrophoresis; CPY, yeast carboxypeptidase Y.     

Lightning density and burning frequency in South African vegetation

This study investigates lightning density in relation to burning frequency in five South African biomes. Data from automatic lightning flash counters distributed throughout South Africa are used to estimate the range and mean values of annual lightning ground-strike density in each biome. The lowest average lightning frequencies were recorded in the karoo and fynbos biomes, while the highest values were obtained in the sour and inland mountain divisions of the grassland biome, with intermediate values computed for the savanna-woodland and forest biomes. These results were compared with published findings on the effects of different burning frequencies on plant communities in each biome. In general, plant communities occurring in areas experiencing the highest annual lightning densities (e.g., sour and mixed grassland, and moist savanna-woodland) tolerate and require more frequent burning than those typical of areas subject to lower lightning densities (e.g., fynbos, forest, sweet grassland, and arid savanna-woodland). These findings suggest that the constituent plant populations in each biome have adapted to burning frequency according to the local probability of ignition by lightning in the areas they inhabit, and that present day responses of plant communities to burning reflect their ancestral exposure to fire in the course of their evolutionary development under pre-existing, natural fire regimes.Research sponsered by the South African Council for Scientific and Industrial Research. We thank the Director, National Electrical Engineering Research Institute (NEERI), Pretoria, South Africa, and H. Kröninger, Lightning Research Division, NEERI, for providing data on lightning ground-strike densities and coordinates and elevations for counters in South Africa. We thank Drs J. H. Bock and M. T. Mentis for commenting on an early draft of this paper.     

Comparative mapping in F2∶3 and F6∶7 generations of quantitative trait loci for grain yield and yield components in maize

This study was conducted to compare maize quantitative trait loci (QTL) detection for grain yield and yield components in F₂₃ and F₆₇ recombinant inbred (RI) lines from the same population. One hundred and eighty-six F₆₇ RIs from a Mo17×H99 population were grown in a replicated field experiment and analyzed at 101 loci detected by restriction fragment length polymorphisms (RFLPs). Single-factor analysis of variance was conducted for each locus-trait combination to identify QTL. For grain yield, 6 QTL were detected accounting for 22% of the phenotypic variation. A total of 63 QTL were identified for the seven grain yield components with alleles from both parents contributing to increased trait values. Several genetic regions were associated with more than one trait, indicating possible linked and/or pleiotropic effects. In a comparison with 150 F₂₃ lines from the same population, the same genetic regions and parental effects were detected across generations despite being evaluated under diverse environmental conditions. Some of the QTL detected in the F₂₃ seem to be dissected into multiple, linked QTL in the F₆₇ generation, indicating better genetic resolution for QTL detection with RIs. Also, genetic effects at QTL are smaller in the F₆₇ generation for all traits.Abbreviations RFLPs Restriction fragment length polymorphisms - QTL quantitative trait loci - RIs recombinant inbreds Journal Paper no. J-16261 of the Iowa Agric and Home Economics Exp Stn Project no. 3134     

RFLP markers to identify the alleles on the Mla locus conferring powdery mildew resistance in barley

Summary To identify the mildew resistance locus Mla in barley with molecular markers, closely linked genomic RFLP clones were selected with the help of near-isogenic lines having the Pallas and Siri background. Out of 22 polymorphic clones 3 were located around the Mla locus on chromosome 5 with a distance of 5.1 + 2.9 cM (MWG 1H068), 4.2±1.7 cM (MWG 1H060) and 0.7 ± 0.7 cM (MWG 1H036), respectively. The polymorphic clone MWG 1H036 displayed the same RFLP pattern in both Pallas and Siri near-isogenic lines and in different varieties digested with six restriction enzymes possessing the same mildew resistance gene. The alleles of the Mla locus were grouped in 11 classes according to their specific RFLP patterns; 3 of these groups contain the majority of Mla alleles already used in barley breeding programs in Europe.     

Lipooligosacc haridic antigens fromMycobacterium kansasii andMycobacterium gastri

A set of Lipooligosaccharides (LOSs) has previously been characterized inM. gastri W471. The structure of the highly antigenic LOS (LOS-III) was elucidated and this molecule can unambiguously distinguishM. gastri from the opportunistic pathogenM. kansasii. In the present study, the structures of three otherM. gastri W471 LOSs were determined by one-dimensional¹H-NMR spectroscopy and gas liquid chromatography. They differ by the number of Xylp units and by the structure of the distal monosaccharide. The two dimensional (2D) NMR approach was successfully applied to the LOS antigen ofM. kansasii to locate the acetyl and acyl substituents and to determine the anomeric configuration of the -d-Fucp unit.The molecular specificity of anti-LOS-III antibodies was investigated and the LOS-III epitope was defined as the distal disaccharide: 3,6-dideoxy-4-C-(1,3-dimethoxy-4,5,6,7-tetrahydroxy-heptyl)--xylohexp-(1 3)--l-Xylp.Abbreviations CI Chemical Ionisation - COSY ¹H-¹H COrrelated SpectroscopY - COSY LR ¹H-¹H COrrelated SpectroscopY Long Range - DQF-COSY Double Quantum Filtered¹H-¹H COrrelated SpectroscopY - EI Electronic Impact - GC Gas Chromatography - HMBC Heteronuclear Multiple Bond Correlation spectroscopy - HMQC Heteronuclear Multiple Quantum Correlation spectroscopy - HOHAHA HOmonuclear HArtmann-HAhn spectroscopy - HPLC High Performance Liquid Chromatography - ³J_H.H vicinal spin-spin coupling constants - LAM LipoArabinoMannan - LOS LipoOligoSaccharide - MS Mass Spectrometry - FAB/MS Fast Atom Bombardment-Mass Spectrometry - ¹H- or¹³C- NMR proton or carbon Nuclear Magnetic Resonance - PBS Phosphate Buffered Saline - PheG1 K-I Major phenolic glycolipids fromM. kansasii - RCT-1, -2, -3 relayed coherence transfer 1 step, 2 steps, 3 steps - ROESY Rotative frame Overhauser Effect SpectroscopY - Sugars Fucp: fucopyranose - Galp galactopyranose - Glcp glucopyranose - hexp hexopyranose - Rhap rhamnopyranose - Xylp xylopyranose - TLC Thin Layer Chromatography - TMS Tri Methyl Silyl     

Selectively 13C-enriched DNA: Evidence from 13C1′ relaxation rate measurements of an internal dynamics sequence effect in the lac operator

Summary In order to study some internal dynamic processes of the lac operator sequence, the ¹³C-labeled duplex 5d(C₀G₁C₂T₃C₄A₅C₆A₇A₈T₉T₁₀) · d(A₁₀A₉T₈T₇G₆T₅G₄A₃G₂C₁G₀)3 was used. The spreading of both the H1 and C1 resonances brought about an excellent dispersion of the ¹H1-¹³C1 correlations. The spinlattice relaxation parameters R(C_z), R(C_x,y) and R(H_zC_z) were measured for each residue of the two complementary strands, except for the 3-terminal residues which were not labeled. Variation of the relaxation rates was found along the sequence. These data were analyzed in the context of the model-free formalism proposed by Lipari and Szabo [(1982) J. Am. Chem. Soc., 104, 4546–4570] and extended to three parameters by Clore et al. [(1990) Biochemistry, 29, 7387–7401; and (1990) J. Am. Chem. Soc., 112, 4989–4991]. A careful analysis using a least-squares program showed that our data must be interpreted in terms of a three-parameter spectral density function. With this approach, the global correlation time was found to be the same for each residue. All the C1-H1 fragments exhibited both slow (_s = 1.5) and fast (_f = 20 ps) restricted libration motions (S _inf2 ^sups =0.74 to 1.0 and S _inf2 ^supf =0.52 to 0.96). Relaxation processes were described as governed by the motion of the sugar relative to the base and in terms of bending of the whole duplex. The possible role played by the special structure of the AATT sequence is discussed. No evident correlation was found between the amplitude motions of the complementary residues. The 5-terminal residues showed large internal motions (S²=0.5), which describe the fraying of the double helix. Global examination of the microdynamical parameters S _inf2 ^supf and S _inf2 ^sups along the nucleotide sequence showed that the adenine residues exhibit more restricted fast internal motions (S _inf2 ^supf =0.88 to 0.96) than the others, whereas the measured relaxation rates of the four nucleosides in solution were mainly of dipolar origin. Moreover, the fit of both R(C_z) and R(HzC_z) experimental relaxation rates using an only global correlation time for all the residues, gave evidence of a supplementary relaxation pathway affecting R(C_x,y) for the purine residues in the (53) G₄A₃ and A₁₀A₉T₈T₇ sequences. This relaxation process was analyzed in terms of exchange stemming from motions of the sugar around the glycosidic bond on the millisecond time scale. It should be pointed out that these residues gave evidence of close contacts with the protein in the complex with the lac operator [Boelens et al. (1987) J. Mol. Biol., 193, 213–216] and that these motions could be implied in the lac-operator-lac-repressor recognition process.