l-phenylalanine production by double auxotrophic multianalogue-resistant mutant ofArthrobacter globiformis

Tryptophan-plus-tyrosine double auxotrophic mutants resistant to fluorophenylalanine (PFP) and β-2-thienylalanine (TA) were isolated from a biotin-requiring glutamate-producingArthrobacter globiformis. The mutants were found to producel-phenylalanine in mineral salts medium. Further improvement ofl-phenylalanine production was achieved by isolation of mutants resistant to 5-methyltryptophan (MT) and 3-nitrotyrosine (NT) from a double auxotrophic PFP^r and TA^r mutant. Under optimal cultural condition one mutant yielded 9.6g phenylalanine per L medium in flask culture. Enzymic activity of regulatory enzymes (deoxy-d-arabino-heptulosonate-7-phosphate synthase, chorismate mutase and prephenate dehydratase) were observed in the wild type, double auxotroph and double-auxotrophic multianalogue-resistant mutant.     

Structural studies of the glycopeptides of B-chain of cinnamomin – a type II ribosome-inactivating protein by nuclear magnetic resonance

Cinnamomin is a plant type II ribosome-inactivating protein (RIP) isolated from the seeds of Cinnamomum camphora. It consists of two nonidentical polypeptide chains (A- and B-chain) held together through one disulfide linkage. Its A- and B-chain contain 0.3% and 3.9% sugars respectively. The B-chain of cinnamomin was digested by pronase E and then the liberated glycopeptides were separated from non-glycopeptides by gel filtration chromatography on a Bio-Gel P-4 column. Three crude glycopeptides were obtained by continuing chromatography over anion-exchange resin (AG1-X2) in the buffer of 2% pyridine-acetic acid (pH 8.3) with a polygradient elution system. Through further purification by the gel filtration chromatography and HPLC, three major glycopeptides, GP1, GP2 and GP3 were obtained. Mainly by two-dimensional Nuclear Magnetic Resonance (NMR) including TOCSY, DQF-COSY, NOESY, HMQC and HMBC, their primary structures were analyzed as: Man1,3Man1,6(Man1,3)(Xyl1,2)Man1,4GlcNAc1,4GlcNAc1-(Gly-)Asn-Asn-Thr(GP1), Man1,6(Man1,3)(Xyl1,2)Man1,4GlcNAc1,4(Fuc1,3)GlcNAc1-Asn-Ala-Thr(GP2),Man1,6(Man1,3)Man1,6(Man1,2 Man1,3)Man1,4GlcNAc1,4GlcNAc1-(Ala-)Asn-Gly-Thr(GP3).     

The solution structure of rat Aβ-(1–28) and its interaction with zinc ion: insights into the scarcity of amyloid deposition in aged rat brain

The amyloid -peptide (A) is a major component of insoluble amyloid deposits in Alzheimers disease, and the ability of the -peptide to exist in different conformations is dependent on residues 1–28 [-(1–28)]. However, different from humans, no A amyloid deposition has been found in aged rats brains. Studying the three-dimensional solution structure of rat A-(1–28) and the binding circumstance of Zn²⁺ is beneficial to a clear understanding of the potential role of Zn²⁺ in Alzheimer-associated neuropathogenesis and to suggest why there is no amyloid deposition in aged rats brains. Here we used nuclear magnetic resonance (NMR) spectroscopy to determine the solution structure of rat A-(1–28) and the binding constant of Zn²⁺ to rat A-(1–28). Our results suggest that (1) the three-dimensional solution structure of rat A-(1–28) is more stable than that of human A-(1–28) in DMSO-d₆ and that a helical region from Glu16 to Val24 exists in the rat A-(1–28); (2) the affinity of Zn²⁺ for rat A-(1–28) is lower than that for human A-(1–28) and the NMR data suggest that Arg13, His6, and His14 residues provide the primary binding sites for Zn²⁺; and (3) the proper binding of Zn²⁺ to rat A-(1–28) can induce the peptide to change to a more stable conformation.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00775-004-0556-xAbbreviations A amyloid -peptide - AD Alzheimers disease - hA-(1–28) human A-(1–28) - rA-(1-28) rat A-(1–28) - REM restrained energy minimization     

Degradation of the diarylpropane lignin model compound 1-(3′,4′-diethoxyphenyl)-1,3-dihydroxy-2-(4′'-methoxyphenyl)-propane and derivatives by the basidiomycete Phanerochaete chrysosporium

The white rot basidiomycete Phanerochaete chrysosporium metabolized 1-(3,4-diethoxyphenyl)-1,3(dihydroxy)-2-(4'-methoxyphenyl)-propane (XII) in low nitrogen stationary cultures, conditions under which the ligninolytic enzyme system is expressed. 3,4-Diethoxybenzyl alcohol (IV), 1,2(dihydroxy)-1-(4-methoxyphenyl)ethane (XX) and anisyl alcohol were isolated as metabolic products indicating an initial , bond cleavage of this dimer. Exogenously added XX was rapidly converted to anisyl alcohol, indicating that XX is an intermediate in the metabolism of XII. Fungal cleavage of the , bond of 1-(3-4-diethoxyphenyl)-1-(hydroxy)-2-(4'-methoxyphenyl)ethane (XI) also occurred, indicating that a hydroxymethyl group is not a prerequisite for this reaction. P. chrysosporium also metabolized 1-(4-ethoxy-3-methoxyphenyl)-2,2(dihydroxy)-2-(4'-methoxyphenyl)propane-1-ol (XIII). The major products of the degradation of this triol included 4-ethoxy-3-methoxybenzyl alcohol (III) and 2-hydroxy-1-(4-methoxyphenyl)-1-oxoethane (XXI). The nature of the products formed indicates that this triol is also cleaved directly at the , bond. The significant difference in the nature of the products formed from the diaryl propane (XII) and the triol (XIII), however, suggests that XIII is not an intermediate in the major pathway for the degradation of XII. Metabolites were identified after comparison with chemically synthesized standards by GLC-mass spectrometry.Abbreviations GLC Gas liquid chromatography - TMSi trimethylsilyl - TLC thin layer chromatography - MS mass spectrometry     

First copulation increases longevity and fecundity of <Emphasis Type="Italic">Histiostoma feroniarum</Emphasis> (Acari: Astigmata: Acaridida) females

I investigated the influence of insemination at different life stages on female fitness in the mite Histiostoma feroniarum. In this species, males guard immature females at the tritonymph stage to inseminate them immediately after the last moulting. Four groups of females were studied (1) females inseminated naturally, i.e. mating occurred immediately after guarding, and then the male was removed (IF/0M), (2) naturally inseminated females, where after insemination the male was replaced by two additional males (IF/2M), (3) virgin females reared without males (VF/0M) and (4) mature, virgin females to which two virgin males were added 3 days after last moulting (VF/2DM). The results show that females inseminated naturally (IF/0M) have higher longevity and fecundity than either virgin (VF/0M) or late-inseminated females (VF/2DM). Furthermore, longevity and fecundity of the former (IF/0M) was also greater than that of females naturally inseminated and subsequently exposed to males (IF/2M). One may suggest that seminal fluids have a positive effect on female fitness. When delayed insemination occurs, such positive effect may not be observed due to a change in features of the sperm access system. Harassment may explain decreased longevity and fecundity of females inseminated naturally compared to females that received additional males.This revised version was published online in May 2005 with a corrected cover date.     

Structure of triphosphoryl nucleotide bound at the active site of yeast hexokinase: 1H-nuclear magnetic resonance study

Conformation of a nonhydrolyzable adenosine triphosphate (ATP) analogue, adenylyl-(,-methylene)-diphosphonate (AMPPCP) bound at the active site of yeast hexokinase-PII was determined by proton two-dimensional transferred nuclear Overhauser effect spectroscopy (TRNOESY) and molecular dynamics simulations. The effect of the glucose-induced domain closure on the conformation of the nucleotide was evaluated by making measurements on two different complexes: PIIAMPPCPMg(II) and PIIGlcAMPPCPMg(II). TRNOE measurements were made at 500 MHz, 10°C, as a function of several mixing times varying in the range of 40 to 200 ms. Interproton distances derived from the analysis of NOE buildup curves were used as restraints in molecular dynamics simulations to determine the conformation of the enzyme bound nucleotide. The adenosine moiety was found to bind in high anti conformation with a glycosidic torsion angle = 48 ± 5 degrees in both complexes. However, significant differences in the conformations of the ribose and triphosphoryl chain of the nucleotide are observed between the two complexes. The phase angles of pseudorotation P in PIIAMPPCPMg(II) and PIIGlcAMPPCPMg(II) are 87 degrees and 77 degrees, describing a OE and OT₄ sugar pucker and the amplitudes of the sugar pucker () are 37 degrees and 61 degrees, respectively.     

Alkoxy-substituted group 6 allenylidene complexes - Synthesis and properties

Bis(alkoxy)allenylidene complexes, [(CO)₅MCCC(OR′)OR], as well as mono(alkoxy)allenylidene complexes, [(CO)₅MCCC(OR′)Ph], of chromium and tungsten are accessible from propynones [HCCC(O)Ph] or propynoic acid esters [HCCC(O)OR; R = Et, (−)-menthyl, endo-bornyl] by the following reaction sequence: (a) deprotonation of the alkynes, (b) reaction with [(CO)₅M-THF] (M = Cr, W), and (c) alkylation of the resulting alkynyl metallate, [(CO)₅MCCC(O)R], with Meerwein salts. Vinylidene complexes, [(CO)₅MCC(R′)C(O)OR], are formed as a by-product by C_β-alkylation of the alkynyl metallate. Dimethylamine displaces one alkoxy substituent of the bis(alkoxy)allenylidene complexes to give dimethylamino(alkoxy)allenylidene complexes, [(CO)₅MCCC(OR)NMe₂]. The analogous reaction of dimethylamine with a mono(alkoxy)-substituted allenylidene complex affords the aminoallenylidene complex [(CO)₅CrCCC(NMe₂)Ph]. When the amine is used in large excess, the α,β-unsaturated aminocarbene complex [(CO)₅CrC(NMe₂)C(H)C(NMe₂)Ph] is additionally formed by addition of the amine across the C_αC_β-bond of the allenylidene ligand. The reaction of [(CO)₅MCCC(OEt)₂] with dimethyl ethylenediamine offers access to bis(amino)allenylidene complexes, in which C_γ is part of a five-membered heterocycle. Photolysis of bis(alkoxy)allenylidene complexes in the presence of triphenylphosphine yields tetracarbonyl- and tricarbonyl{bis(phosphine)}allenylidene complexes. Diethylaminopropyne inserts into the C_βC_γ bond of [(CO)₅MCCC(OEt)OMethyl] to give alkenylallenylidene complexes. Subsequent acid-catalyzed intramolecular cyclization affords a pyranylidene complex.     

Detection of plum pox virus in leaves and aphids by SIBA and DAS-ELISA assays

The slot-immunobinding assay (SIBA) was adapted for detection of plum pox virus (PPV) and compared with DAS-ELISA. SIBA was easy to perform and as sensitive as DAS-ELISA in detection of various PPV isolates in herbaceous and woody plants, but not in aphids (Myzus persicae).     

Microbial degradation of dehydrodiconiferyl alcohol,a lignin substructure model

Bacteria, yeasts, and molds which grew in a medium containing a synthetic lignin — a dehydrogenation polymer (DHP) of coniferyl alcohol — as a sole carbon source, were isolated from soil. One fungus, Fusarium solani M-13-1, was found to degrade the DHP most vigorously among the isolated organisms. It was shake-cultured in a medium containing dehydrodiconiferyl alcohol (DHCA) (I), an important lignin model compound, and the following six metabolic products were isolated and identified: 1) Phenylcoumaran--aldehydic (II) and -carboxylic compounds, 2) phenylcoumaran--aldehydic compound (IV), formed by release of a 2-carbon fragment from the phenylcoumaran--carboxylic compound, 3) 5-acetylvanillyl alcohol (V), formed by cleavage of the coumaran ring and reduction of the -aldehyde group, 4) 5-carboxyvanillyl alcohol (VI), formed by subsequent oxidation of the acetyl group, and 5) the -ether of DHCA (VII), considered to be a by-product. A degradation pathway for DHCA was proposed on the basis of these metabolic products.Non-Standard Abbreviations DHP dehydrogenation polymer - DHCA dehydrodiconiferyl alcohol - DDQ dichlorodicyano-p-benzoquinone - DDHQ dichlorodicyano-p-hydroquinone - Ar aromatic - TLC thin layer chromatography - GC-MS gas chromatography-mass spectrometry     

Location of a gene regulating drought-induced abscisic acid production on the long arm of chromosome 5A of wheat

The accumulation of abscisic acid (ABA) by detached and partially dehydrated wheat leaves is known to be inherited in a quantitative manner. The location of genes having a major effect on drought-induced ABA accumulation in wheat was determined using a set of single chromosome substitution lines and populations derived from a cross between a high-ABA- and a low-ABA-producing genotype. Examination of a series of chromosome substitution lines of the high-ABA genotype Ciano 67 into the low-ABA recipient Chinese Spring showed that chromosome 5A carries gene(s) that have a major influence on ABA accumulation in a drought test with detached and partially dehydrated leaves (DLT). A similar DLT was used to examine ABA accumulation in a population of F₂ plants and doubled haploid (DH) lines derived from the cross between Chinese Spring (low-ABA) and SQ1 (high-ABA) in which the F₂ population (139 plants) and DH lines (96 lines) were also mapped partially with molecular markers. Analysis of variance of ABA accumulation between and within marker allele classes in the F₂ confirmed the location of a gene(s) regulating ABA accumulation on the long arm of chromosome 5A. MAPMAKERQTL showed the most likely position for the ABA quantitative trait locus (QTL) to be between the loci Xpsr575 and Xpsr426, about 8 cM from Xpsr426. A similar trend for high ABA accumulation was found in DH lines having the SQ1 allele at marker loci in the same region of chromosome 5AL, but the QTL effect was not significant. The function of the QTL is discussed.     

Altered GABAergic and glutamatergic transmission in audiogenic seizure-susceptible mice

The C57BL/10 SPS/sps mouse mutant are audiogenic seizure-susceptible. The enzymatic activities of glutamate decarboxylase (GAD), GABA aminotransferase (GABA-T), alanine aminotransferase (ALA-T), aspartate aminotransferase (ASP-T), and glutamate dehydrogenase (GDH) of whole brain supernatant are significantly reduced in these epileptic mice. GABA uptake is decreased in cortex, midbrain, and pons medulla. Previous studies showed the presence of two sodium-dependent GLU uptake systems in normal (SPS/SPS) mice. Glutamate U_max by System 1 is significantly decreased in these mice, whereas the U_max value for System 2 is significantly increased in the epileptic mice.     

Conformational analysis of a Chlamydia-specific disaccharide α-Kdo-(2→8)-α-Kdo-(2→O)-allyl in aqueous solution and bound to a monoclonal antibody: Observation of intermolecular transfer NOEs

The disaccharide -Kdo-(28)--Kdo (Kdo: 3-deoxy-d-manno-oct-2-ulosonic acid) represents a genus-specific epitope of the lipopolysaccharide of the obligate intracellular human pathogen Chlamydia. The conformation of the synthetically derived disaccharide -Kdo-(28)--Kdo-(2O)-allyl was studied in aqueous solution, and complexed to a monoclonal antibody S25-2. Various NMR experiments based on the detection of NOEs (or transfer NOEs) and ROEs (or transfer ROEs) were performed. A major problem was the extensive overlap of almost all ¹H NMR signals of -Kdo-(28)--Kdo-(2O)-allyl. To overcome this difficulty, HMQC-NOESY and HMQC-trNOESY experiments were employed. Spin diffusion effects were identified using trROESY experiments, QUIET-trNOESY experiments and MINSY experiments. It was found that protein protons contribute to the observed spin diffusion effects. At 800 MHz, intermolecular trNOEs were observed between ligand protons and aromatic protons in the antibody binding site. From NMR experiments and Metropolis Monte Carlo simulations, it was concluded that -Kdo-(28)--Kdo-(2O)-allyl in aqueous solution exists as a complex conformational mixture. Upon binding to the monoclonal antibody S25-2, only a limited range of conformations is available to -Kdo-(28)--Kdo-(2O)-allyl. These possible bound conformations were derived from a distance geometry analysis using transfer NOEs as experimental constraints. It is clear that a conformation is selected which lies within a part of the conformational space that is highly populated in solution. This conformational space also includes the conformation found in the crystal structure. Our results provide a basis for modeling studies of the antibody–disaccharide complex.     

Purification of (1→3)-β-glucan endohydrolase isoenzyme II from germinated barley and determination of its primary structure from a cDNA clone

A (13)--D-glucan 3-glucanonydrolase (EC 3.2.1.39) of apparent M _r 32 000, designated GII, has been purified from germinated barley grain and characterized. The isoenzyme is resolved from a previously purified isoenzyme (GI) on the basis of differences in their isoelectric points; (13)--glucanases GI and GII have pI values of 8.6 and 10.0, respectively. Comparison of the sequences of their 40 NH₂-terminal amino acids reveals 68% positional identity. A 1265 nucleotide pair cDNA encoding (13)--glucanase isoenzyme GII has been isolated from a library prepared with mRNA of 2-day germinated barley scutella. Nucleotide sequence analysis of the cDNA has enabled the complete primary structure of the 306 amino acid (13)--glucanase to be deduced, together with that of a putative NH₂-terminal signal peptide of 28 amino acid residues. The (13)--glucanase cDNA is characterized by a high (G+C) content, which reflects a strong bias for the use of G or C in the wobble base position of codons. The amino acid sequence of the (13)--glucanase shows highly conserved internal domains and 52% overall positional identity with barley (13, 14)--glucanase isoenzyme EII, an enzyme of related but quite distinct substrate specificity. Thus, the (13)--glucanases, which may provide a degree of protection against microbial invasion of germinated barley grain through their ability to degrade fungal cell wall polysaccharides, appear to share a common evolutionary origin with the (13, 14)--glucanases, which function to depolymerize endosperm cell walls in the germinated grain.     

Evidence of regional differences in the lectin histochemistry along the ductus epididymis of the lizard,Podarcis sicula Raf

The regional difference in the carbohydrate components of the ductus epididymis epithelium of a lizard was delineated by means of 13 lectins. Basal cells expressed only N-acetylglucosamine (GlcNAc). Throughout the ductus, the secretory cells showed oligosaccharides with terminal N-acetylneuraminic acid (Neu5Ac)(2,6)galactose (Gal)/N-acetylgalactosamine (GalNAc) and internal mannose (Man) and/or glucose (Glc) in the whole cytoplasm, oligosaccharides terminating in Neu5Ac(2,6)Gal(1,3)GalNAc, Neu5Ac(2,6)Gal (1,4)GlcNAc, GalNAc, GlcNAc, and fucose (Fuc) in the supra-nuclear zone, and also glycans terminating in Neu5Ac(2,3)Gal (1,4)GlcNAc, Neu5Ac(2,6)Gal(1,3)GalNAc, Gal (1,4)GlcNAc on the luminal surface. In the caput and corpus regions, the supra-nuclear cytoplasm was characterized by terminal Gal(1,4)GlcNAc and GalNAc, the luminal surface by GalNAc and Gal. The Golgi zone, showing oligosaccharides with terminal Neu5Ac(2,3)Gal (1,4)GlcNAc, Neu5Ac(2,6)Gal (1,3)GalNAc, Neu5Ac(2,6)Gal (1,4)GlcNAc, and internal GlcNAc, expressed terminal Gal (1,4)GlcNAc and GalNAc in the caput, and terminal GalNAc in the corpus. The granules showed all the investigated carbohydrates in their peripheral zone except terminal GalNAc and Fuc, whereas internal Man/Glc and terminal Gal were expressed in the central core, and Fuc throughout the ductus, terminal GlcNAc in the caput and corpus, and terminal GalNAc only in the corpus.     

Structure-Based Rational Design of a Phosphotriesterase

In silico substrate docking of both stereoisomers of the pesticide chlorfenvinphos (CVP) in the phosphotriesterase from Agrobacterium radiobacter identified two residues (F131 and W132) that prevent productive substrate binding and cause stereospecificity. A variant (W131H/F132A) was designed that exhibited ca. 480-fold and 8-fold increases in the rate of Z-CVP and E-CVP hydrolysis, respectively, eliminating stereospecificity.Synthetic organophosphate pesticides (OPs) can cause acute neurotoxicity in insects and humans as a result of their inhibition of acetylcholinesterase at the nerve synapse (15). The >90% identical bacterial phosphotriesterases (PTEs) from Pseudomonas diminuta (oph; PTE_Pd) (5) and Agrobacterium radiobacter (opdA; PTE_Ar) (9) efficiently catalyze the hydrolysis of a broad range of OPs, effectively detoxifying them. This has led to the commercialization of PTE_Ar as a free-enzyme bioremediant (14) and its use in treating OP poisoning in animal studies (1). However, not all OPs are efficiently turned over by the PTEs. For instance, despite having a reasonably reactive leaving group (Fig. ​(Fig.1),1), the turnover of chlorfenvinphos (CVP) by PTE_Ar was not detected in a previous study (9).Open in a separate windowFIG. 1.Structures of the leaving groups of all the substrates discussed in this work. (a) 3,5,6-Trichloro-2-pyridinol for methyl chlorpyrifos oxon; (b) 4-nitrophenol for methyl paraoxon and methyl parathion; (c) 2,2-dichloroethenol for dichlorvos; (d) Z/E-2-chloro-1-(2,4-dichlorophenyl)ethanol for E/Z-CVP; (e) 4-methoxyphenol for EPO.     

Brain Net Unidirectional Uptake of α-[14C]Methyl-L-Tryptophan (α-MTrp) and Its Correlation with Regional Serotonin Synthesis,Tryptophan Incorporation into Proteins,and Permeability Surface Area Products of Tryptophan and α-MTrp

The uptake and trapping constants for labeled tryptophan (Trp) via the serotonin (5-hydroxytryptamine; 5-HT) metabolic pathway and for the incorporation of Trp into proteins, and -[¹⁴C]methyl-L-tryptophan (-MTrp) were measured. Measurements were done in rats treated with either saline or probenecid (200 mg/kg). In addition, the blood-brain barrier (BBB) permeability surface area products for Trp (PS_T) and -MTrp (PS) were measured in normal rats. The results suggest that, in both groups of rats, there is a highly significant correlation (p < 0.05; Pearson Product Moment Correlation (PPMC) between the brain uptake and trapping constants for -MTrp and those of Trp via the 5-HT metabolic pathway, but there is no significant correlation (p > 0.05; PPMC) between either of these constants and the PS products of either compound. There is also no significant correlation (p > 0.05; PPMC) between the constant for the Trp incorporation into proteins with any of the other parameters. For all parameters, except Trp incorporation into proteins (-MTrp is not incorporated into proteins), there was a highly significant correlation (p < 0.001) between the quantities measured for Trp and -MTrp. The data presented here strongly suggests that the brain uptake and trapping of -MTrp relates to brain 5-HT synthesis, and does not relate to the BBB transport or protein incorporation of Trp. On the basis of these results, as well as those previously reported, we concluded that trapping (unidirectional uptake) of -MTrp can be converted to the 5-HT synthesis rates in the brain. From this also follows that labeled -MTrp is a good tracer for in vivo evaluation of the brain 5-HT synthesis.     

Hormone-like product of vascular tissue stimulating starch accumulation in pith explants of kale and endogenous cytokinins

When stem explants of kale (Brassica oleracea L. var.medullosa), containing pith parenchyma and a strip of vascular tissue, were cultured on simple sucrose medium, a hormone-like factor was transported from the vascular tissue to the adjacent pith, where it stimulated accumulation of starch. Similarly, up to a sevenfold increase of starch content in explants could be induced by cytokinins added to the culture medium. The relative stimulatory effect of several cytokinins (5×10^?6 M) and hormone-like product of vascular tissue (HPVT) in a typical experiment were: control (1.0), trans-zeatin (6.7), HPVT (6.2), N⁶-[2-isopentenyl]adenine (5.4), transzeatin riboside (5.2), N⁶-[2-isopentenyl]adenosine (5.4), kinetin (3.6), 6-benzylaminopurine (3.5), and adenine (2.1). Concentration of endogenous cytokinins was determined using ELISA (trans-zeatin, N⁶-[2-isopentenyl]adenine and their ribosides) andAmaranthus bioassay (total cytokinins). No effect of vascular tissue on the level of endogenous cytokinins in explants was found. The results support the conclusions of previous experiments that the HPVT stimulating starch accumulation is not a cytokinin.     

A neural coding model for sensory intensity discrimination,to be applied to gustation

Summary This paper presents a model of the neural coding and discrimination of sensory intensity. The model consists of five stages: (1) the coding of stimulus intensity in peripheral receptors or neurons by a rate code. The relevance of comparing different analysis intervals for the response is pointed out; (2) neural processing, according to either labeled-line or across-fiber pattern theory. In addition, two possible non-linearities in the processing are considered: a threshold mechanism, and contrast enhancement by reciprocal inhibition; (3) a neural discriminator, based on signal-detection theory; (4) a memory stage; (5) an effector organ providing a behavioral output. Emphasis is put on stages 2 and 3.The model produces predictions of the differential threshold, which should be directly testable in a behavioral two-alternative forced-choice paradigm. The model will be applied to gustatory intensity discrimination in rat in a subsequent study (Maes and Erickson 1984). The Discussion pays attention to the relative contributions of peripheral and central noise sources. It also compares the present model with Beidler's (1958) approach through just noticeable differences (JND's). The model presented here seems more adequate in providing an understanding of sensory information processing.Abbreviations AFP across fiber pattern - DA discrimination acuity - DT differential threshold - JND just noticeable difference - LL labeled line - NTS nucleus tractus solitarius     

Biosynthesis of (1,3)(1,4)-β-glucan and (1,3)-β-glucan in barley (Hordeum vulgare L.)

Mixed membrane preparations from the coleoptiles and first leaves of young barley (Hordeum vulgare L. cv. Triumph) plants catalysed the synthesis of 55% methanol-insoluble labelled material from UDP[U-¹⁴C]glucose, the main components of which were identified as (1,3)(1,4)-- and (1,3)--D-glucans. The membrane preparations also catalysed the transformation of UDP-glucose into labelled low-molecular-weight products, mainly glucose (by phosphatase action), glucose-1-phosphate (by phosphodiesterase action) and glyco(phospho)lipids (by glycosyltransferase action). The formation of (1,3)(1,4)--glucans, (1,3)--glucans, and the other reactions competing for UDP-glucose, were monitored simultaneously and quantitatively by a novel procedure based on enzymatic analysis, thin-layer chromatography and digital autoradiography. Thus it was possible (i) to optimise conditions to obtain (1,3)(1,4)--glucan synthesis or (1,3)--glucan synthesis in isolation, and (ii) to study the influence of temperature, pH, cofactors, substrate concentration etc. on the (1,3)(1,4) and (1,3)--glucan synthesis reactions even when both occurred together. The synthesis of both -glucans was optimal at 20°C. In Tris-HCl buffer, the pH optima for (1,3)(1,4)--glucan synthesis and (1,3)--glucan synthesis were pH 8.5 and pH 7.0, respectively. Both glucan-synthesis reactions required Mg²⁺: (1,3)--glucan synthesis was optimal at 2 mM, whereas (1,3)(1,4)--glucan synthesis continued to increase up to 200 mM Mg²⁺, when the ion was supplied as the sulphate. (1,3)--Glucan synthesis was Ca²⁺ dependent and this dependence could be abolished by proteinase treatment. The K _m with respect to UDP-glucose was 1.5 mM for (1,3)--glucan synthesis and approximately 1 mM for (1,3)(1,4)--glucan synthesis. The (1,3)(1,4)--glucan formed in vitro had the same ratio of trisaccharide to tetrasaccharide structural blocks irrespective of the experimental conditions used during the synthesis: its enzymatic fragmentation pattern was indistinguishable from that of barley endosperm (1,3)(1,4)--glucan. This indicates either a single synthase enzyme, which is responsible for the formation of both linkage types, or two enzymes which are very tightly coupled functionally.Abbreviations G4G4G3G Glc(1,4)Glc(1,4)Glc(1,3)Glc (-linked) - UDP-Glc uridine-5-diphosphate glucose We are grateful to the Commission of the European Communities for the award of Training Fellowships to Christine Vincent and Martin Becker.     

Efflux of hydraulically lifted water from mycorrhizal fungal hyphae during imposed drought

Apart from improving plant and soil water status during drought, it has been suggested that hydraulic lift (HL) could enhance plant nutrient capture through the flow of mineral nutrients directly from the soil to plant roots, or by maintaining the functioning of mycorrhizal fungi. We evaluated the extent to which the diel cycle of water availability created by HL covaries with the efflux of HL water from the tips of extramatrical (external) mycorrhizal hyphae, and the possible effects on biogeochemical processes. Phenotypic mycorrhizal fungal variables, such as total and live hyphal lengths, were positively correlated with HL efflux from hyphae, soil water potential (dawn), and plant response variables (foliar ¹⁵N). The efflux of HL water from hyphae was also correlated with bacterial abundance and soil enzyme activity (P), and the moistening of soil organic matter. Such findings indicate that the efflux of HL water from the external mycorrhizal mycelia may be a complementary explanation for plant nutrient acquisition and survival during drought.Key words: hydraulic lift, nitrogen, phosphorus, microbial abundance, mycorrhizal hyphae, QuercusIn environments that experience seasonal or extended drought, plant productivity, resource partitioning, and competition are limited by the availability of water and mineral nutrients. One mechanism that is important to whole plant water balance in these environments is hydraulic lift (HL), a passive process driven by gradients in water potential among soils layers. Soil water is transported upwards from deep moist soils and released into the nutrient-rich upper soil layers by root systems accessing both deep and shallow soil layers.¹ HL water may improve the lifespan and activity of fine roots in a wide variety of plant life forms.²Hydraulic lift may also have a second ecological function in facilitating plant nutrient acquisition.² It been hypothesized that HL water could enhance the supply of nutrients to roots through mass flow or diffusion,³ or trigger episodes of soil biotic activity such as microbe-mediated nutrient transformations^4,5 that are analogous to the increased inflow of nitrogen (N) into roots and flushes of carbon (C) and N mineralization respectively that follow precipitation events.^4,6 However, few data currently exist with which to test these possibilities.Hydraulically lifted water also sustains mycorrhizal fungi,^7,8 a mutualism that enhances the acquisition of water and mineral nutrients in many terrestrial plant species. Mycorrhizal fungal hyphae provide comprehensive exploration and rapid access to small-scale or temporary nutrient flushes that may not be available to plant roots.⁹ This resource flow has often been assumed to be a unidirectional flux whereby resources are moved from source (soil) into the sink (plant) by the fungal hyphae. However, there is now evidence to suggest that the physiological plasticity of the peripheral extramatrical hyphae, and in particular the hyphal tips, permits the exudation, and subsequent reabsorption, of water and solutes.^10,11 Laboratory experiments using pure cultures have demonstrated that water may be exuded from the hyphal tips, especially in fungal species with hydrophobic hyphae, along with a variety of organic molecules, such as free amino acids.^10–13 At the same time, water, mobile minerals, amino acids and other low-molecular weight metabolites may be selectively and actively reabsorbed by mycorrhizal fungal hyphae.¹¹ However, quantitative data on the environmental impact of hyphal exudation and reabsorption is still largely lacking.We ask: could the diel cycle of water availability created by HL produce a water efflux from hyphal tips and if so, would this be sufficient to impact biogeochemical processes? Is there also an opposite rhythm driven by plant transpiration so that any resultant soil solution is pulled towards hyphal tips and consequently, the host plant? By imposing drought on seedlings of Quercus agrifolia Nee (coast live oak; Fagaceae) grown in mesocosms (Fig. 1), we identified a composite of feedbacks that could influence nutrient capture with HL (Fig. 2). Our analyses provide support for the key predictions of the HL-nutrient cycling scenario including the efflux of HL water from the extramatrical hyphae (Fig. 3), moistening of soil organic matter (Figs. 3 and ​and4),4), and the maintenance of soil microbial activity and nutrient capture (N, P; Open in a separate windowFigure 1Quercus mesocosms demonstrating the plant, root, and hyphal compartments. Details of soil conditions, plant inoculation protocol, mycorrhizal fungi and dye injection methods are detailed in previous work (ref. 7) Point 1 (tap root compartment) denotes the region in which fluorescent tracer dyes were injected into the mesocosm at dusk to track the path of HL water. Point 2 (hyphal chamber) denotes spots adjacent to or distant from the mesh screen into which a small volume (200 µl) of fluorescent and ¹⁵N tracers (99% as ¹⁵NH₄¹⁵NO₃) were injected at dawn to measure water and nutrient uptake by the external hyphae.Open in a separate windowFigure 2Path analysis of the influence of different soil and mycorrhizal factors on nutrient capture with HL, and resultant model showing the significant path coefficients among variables in the Q. agrifolia mesocosms. Lines with a single arrow denote possible cause-effect relationships. The partial correlation coefficients adjacent to each line indicate the strength of the association between the individual factors. Thick lines are statistically significant (p < 0.05) whereas thin lines indicate no significant relationship between parameters (p > 0.05) and only significant coefficients are given (p < 0.05).Open in a separate windowFigure 3Fluorescently-labeled structures recovered from the hyphal chamber of Quercus microcosms following 80 days of soil drying and with nocturnal hydraulic lift. Yellow-green fluorescence indicates samples labeled with Lucifer yellow CH (LYCH), blue fluorescence denotes samples labeled with Cascade blue (CB) hydrazide. (A) CB-labeled leaf litter from the soil and (B) soil particle; (C) LYCH-labeled root fragment in the soil mixture with adherent extramatrical hyphae; (D) LYCH tracer dye fluorescence in labeled extramatrical hyphae and in efflux (arrow) from the hyphal tip onto organic matter; (E and F) external hyphae filled with LYCH (influx; arrow) and (G) background fluorescence in non-labeled extramatrical hyphae.Open in a separate windowFigure 4Measurements of hyphal efflux and influx based on the quantitative analysis of LYCH fluorescence intensity in soil solution. Fluorescent intensity values were converted to LYCH concentration using a standard curve generated for the dye since fluorescent intensity correlates with the number of fluorescent molecules in solution. Influx is the uptake of LYCH by hyphae as driven by plant transpiration demands (day), and measured efflux is the passive loss of LYCH from hyphae into the surrounding soil during HL (night). Vertical bars indicate the standard error of the means.

Table 1

Summary of soil, microbial, mycorrhizal and plant parameters in plant or hyphal compartments