bluuebunny/crossref_metadata_2025 huggingface dataset:
A novel cycling assay for nicotinic acid–adenine dinucleotide phosphate with nanomolar sensitivity
Authors: GRAEFF Richard , LEE Hon Cheung
Published: September 2002

Nicotinic acid—adenine dinucleotide phosphate (NAADP) is a novel nucleotide derived from NADP that has now been shown to be active in releasing Ca2+ from intracellular stores in a wide variety of cells ranging from plant to human. Despite the obvious importance of monitoring its cellular levels under various physiological conditions, no assay has been reported for NAADP to date. In the present study, a widely applicable assay for NAADP with high sensitivity is described. NAADP was first dephosphorylated to nicotinic acid—adenine dinucleotide by treatment with alkaline phosphatase. The conversion was shown to be stoichiometric. NMN-adenylyltransferase was then used to convert nicotinic acid—adenine dinucleotide into NAD in the presence of high concentrations of NMN. The resultant NAD was amplified by a cycling assay involving alcohol dehydrogenase and diaphorase. Each time NAD cycled through these coupled reactions, a molecule of highly fluorescent resorufin was generated. The reaction could be performed for hours, resulting in more than a 1000-fold amplification. Concentrations of NAADP over the 10—20nM range could be routinely measured. This novel cycling assay was combined with an enzymic treatment to provide the necessary specificity for the assay. NAADP was found to be resistant to NADase and apyrase. Pretreatment of samples with a combination of the hydrolytic enzymes completely eliminated the interference from common nucleotides. The versatility of the cycling assay can also be extended to measure nicotinic acid, which is a substrate in the synthesis of NAADP catalysed by ADP-ribosyl cyclase, over the micromolar range. All the necessary reagents for the cycling assay are widely available and it can be performed using a multi-well fluorescence plate reader, providing a high-throughput method. This is the first assay reported for NAADP and nicotinic acid, which should be valuable in elucidating the messenger functions of NAADP.
Staurosporine induces endothelial cell apoptosis via focal adhesion kinase dephosphorylation and focal adhesion disassembly independent of focal adhesion kinase proteolysis
Authors: KABIR Jahangir , LOBO Melvin , ZACHARY Ian
Published: September 2002

The survival of endothelial cells is dependent on interactions between the matrix and integrins mediated through focal adhesions. Focal adhesion kinase (FAK) is thought to play a key role in maintaining focal adhesion function and cell survival, whereas caspase-mediated FAK proteolysis is implicated in focal adhesion disassembly during apoptosis. We examined the relationship between changes in FAK phosphorylation and proteolysis during apoptosis of primary porcine aortic endothelial cells (PAEC) induced by staurosporine, a widely used apoptogenic agent in diverse cell types. Staurosporine-induced PAEC apoptosis was detected after 1h and was preceded by disruption and loss of FAK localization to focal adhesions within a few minutes, whereas staurosporine-induced cleavage of FAK occurred only after 8—24h. Staurosporine induced a very rapid dephosphorylation of FAK at Tyr861 and Tyr397 and caused dissociation of phosphorylated FAK from focal adhesions as early as 30s. The effect of staurosporine was very potent with striking inhibition of Tyr861 and Tyr397 phosphorylation and focal adhesion disruption occurring in the range 10—100nM. Selective inhibition of a known target of staurosporine, protein kinase C, using GF109203X, and of phosphoinositide 3′-kinase using wortmannin, did not reduce FAK tyrosine phosphorylation at Tyr861 and Tyr397, or cause disruption of focal adhesions. Cycloheximide, the protein synthesis inhibitor, induced PAEC apoptosis more slowly than staurosporine, but did not induce FAK dephosphorylation or rapid focal adhesion disruption, and instead caused a slower loss of focal adhesions and a marked increase in FAK proteolysis. These studies show that FAK dephosphorylation and focal adhesion disassembly are very early events mediating the onset of staurosporine-induced endothelial cell apoptosis and are dissociated from FAK proteolysis. Cycloheximide induces apoptosis through a pathway involving FAK proteolysis without dephosphorylation.
Stopped-flow kinetic studies of electron transfer in the reductase domain of neuronal nitric oxide synthase: re-evaluation of the kinetic mechanism reveals new enzyme intermediates and variation with cytochrome P450 reductase
Authors: KNIGHT Kirsty , SCRUTTON Nigel S.
Published: September 2002

The reduction by NADPH of the FAD and FMN redox centres in the isolated flavin reductase domain of calmodulin-bound rat neuronal nitric oxide synthase (nNOS) has been studied by anaerobic stopped-flow spectroscopy using absorption and fluorescence detection. We show by global analysis of time-dependent photodiode array spectra, single wavelength absorption and NADPH fluorescence studies, that at least four resolvable steps are observed in stopped-flow studies with NADPH and that flavin reduction is reversible. The first reductive step represents the rapid formation of an equilibrium between an NADPH-enzyme charge-transfer species and two-electron-reduced enzyme bound to NADP+. The second and third steps represent further reduction of the enzyme flavins and NADP+ release. The fourth step is attributed to the slow accumulation of an enzyme species that is inferred not to be relevant catalytically in steady-state reactions. Stopped-flow flavin fluorescence studies indicate the presence of slow kinetic phases, the timescales of which correspond to the slow phase observed in absorption and NADPH fluorescence transients. By analogy with stopped-flow studies of cytochrome P450 reductase, we attribute these slow fluorescence and absorption changes to enzyme disproportionation and/or conformational change. Unlike for the functionally related cytochrome P450 reductase, transfer of the first hydride equivalent from NADPH to nNOS reductase does not generate the flavin di-semiquinoid state. This indicates that internal electron transfer is relatively slow and is probably gated by NADP+ release. Release of calmodulin from the nNOS reductase does not affect the kinetics of inter-flavin electron transfer under stopped-flow conditions, although the observed rate of formation of the equilibrium between the NADPH-oxidized enzyme charge-transfer species and two-electron-reduced enzyme bound to NADP+ is modestly slower in calmodulin-depleted enzyme. Our studies indicate the need for significant re-interpretation of published kinetic data for electron transfer in the reductase domain of neuronal nitric oxide synthase.
Determinants of adenophostin A binding to inositol trisphosphate receptors
Authors: MORRIS Stephen A. , NEROU Edmund P. , RILEY Andrew M. , POTTER Barry V.L. , TAYLOR Colin W.
Published: September 2002

Inositol 1,4,5-trisphosphate (IP3) receptors from cerebellum and recombinant type 1 IP3 receptors expressed in Sf9 cells had indistinguishable affinities for IP3 (Kd = 6.40±0.48nM) and adenophostin A (Kd = 0.89±0.05nM). In cytosol-like medium, each of the three mammalian IP3 receptor subtypes when expressed in Sf9 cells bound adenophostin A with greater affinity than IP3. It has been suggested that adenophostin A binds with high affinity only in the presence of ATP, but we found that adenophostin A similarly displaced [3H]IP3 from type 1 IP3 receptors whatever the ATP concentration. N-terminal fragments of the type 1 receptor were expressed with and without the S1 splice site; its removal had no effect on [3H]IP3 binding to the 1—604 protein, but abolished binding to the 224—604 protein. The 1—604 fragment and full-length receptor bound adenophostin A with the same affinity, but the fragment had 3-fold greater affinity for IP3, suggesting that C-terminal residues selectively inhibit IP3 binding. The 224—604S1+ fragment bound IP3 and adenophostin A with increased affinity, but as with the 1—604 fragment it bound adenophostin A with only 2-fold greater affinity than IP3. High-affinity binding of adenophostin A may be partially determined by its 2′-phosphate interacting more effectively than the 1-phosphate of IP3 with residues within the IP3-binding core. This may account for the 2-fold greater affinity of adenophostin A relative to IP3 for the minimal IP3-binding domain. In addition we suggest that C-terminal residues, which impede access of IP3, may selectively interact with adenophostin A to allow it unhindered access to the IP3-binding domain.
A homologue of AMP-activated protein kinase in Drosophila melanogaster is sensitive to AMP and is activated by ATP depletion
Authors: PAN David A. , HARDIE D. Grahame
Published: September 2002

We have identified single genes encoding homologues of the α, β and γ subunits of mammalian AMP-activated protein kinase (AMPK) in the genome of Drosophila melanogaster. Kinase activity could be detected in extracts of a Drosophila cell line using the SAMS peptide, which is a relatively specific substrate for the AMPK/SNF1 kinases in mammals and yeast. Expression of double stranded (ds) RNAs targeted at any of the putative α, β or γ subunits ablated this activity, and abolished expression of the α subunit. The Drosophila kinase (DmAMPK) was activated by AMP in cell-free assays (albeit to a smaller extent than mammalian AMPK), and by stresses that deplete ATP (oligomycin and hypoxia), as well as by carbohydrate deprivation, in intact cells. Using a phosphospecific antibody, we showed that activation was associated with phosphorylation of a threonine residue (Thr-184) within the ‘activation loop’ of the α subunit. We also identified a homologue of acetyl-CoA carboxylase (DmACC) in Drosophila and, using a phosphospecific antibody, showed that the site corresponding to the regulatory AMPK site on the mammalian enzyme became phosphorylated in response to oligomycin or hypoxia. By immunofluorescence microscopy of oligomycin-treated Dmel2 cells using the phosphospecific antibody, the phosphorylated DmAMPK α subunit was mainly detected in the nucleus. Our results show that the AMPK system is highly conserved between insects and mammals. Drosophila cells now represent an attractive system to study this pathway, because of the small, well-defined genome and the ability to ablate expression of specific gene products using interfering dsRNAs.
Regulation of insulin signal transduction pathway by a small-molecule insulin receptor activator
Authors: DING Victor D.H. , QURESHI Sajjad A. , SZALKOWSKI Deborah , LI Zhihua , BIAZZO-ASHNAULT Dawn E. , XIE Dan , LIU Kun , JONES A. Brian , MOLLER David E. , ZHANG Bei B.
Published: September 2002

Insulin regulates cellular metabolism and growth through activation of insulin receptors (IRs). We recently identified a non-peptide small-molecule IR activator (compound 2), which induced human IR tyrosine kinase activity in Chinese-hamster ovary cells expressing human IR [Qureshi, Ding, Li, Szalkowski, Biazzo-Ashnault, Xie, Saperstein, Brady, Huskey, Shen et al. (2000) J. Biol. Chem. 275, 36590—36595]. Oral treatment with this compound resulted in correction of hyperglycaemia, hypertriacylglycerolaemia and hyperinsulinaemia in several rodent models of diabetes. In the present study, we have found that this compound increased tyrosine phosphorylation of the IR β-subunit and IR substrate 1 in primary rat adipocytes as well as induced phosphorylation of Akt, the 70kDa ribosomal protein S6 kinase and glycogen synthase-3 (deactivation) in Chinese-hamster ovary cells expressing human IR. Similar to insulin, compound 2 stimulated glucose uptake, glycogen synthesis and inhibited isoprenaline-stimulated lipolysis in adipocytes. A structurally related analogue (compound 3) was devoid of the above activities suggesting that the activity of compound 2 is specifically mediated by targeted IR activation. The effects of compound 2 on stimulation of glucose uptake, glycogen synthesis and inhibition of lipolysis were blocked by wortmannin, consistent with the involvement of a phosphoinositide 3-kinase-dependent pathway. In addition, compound 2, but not compound 3, exhibited additive or synergistic effects with sub-maximal concentrations of insulin in rat adipocytes. Thus the IR activator was capable of activating insulin-mediated signalling and metabolic pathways in primary adipocytes. These results demonstrate that IR activators have implications for the future development of new therapeutic approaches to Type I and Type II diabetes.
Oligomerization status, with the monomer as active species, defines catalytic efficiency of UDP-glucose pyrophosphorylase
Authors: MARTZ Françoise , WILCZYNSKA Malgorzata , KLECZKOWSKI Leszek A.
Published: September 2002

Barley UDP-glucose pyrophosphorylase (UGPase), a key enzyme for the synthesis of sucrose, cellulose and other saccharides, was expressed in Escherichia coli and purified. Using both native electrophoresis and gel filtration, the recombinant and crude leaf UGPase proteins were found to exist as a mixture of monomers, dimers and higher-order polymers. In order to understand the molecular basis for the oligomerization of UGPase, a conserved Cys residue was replaced (C99S mutant) and several amino acids were substituted (LIV to NIN, KK to LL and LLL to NNN) in a conserved hydrophobic domain (amino acids 117—138). The C99S mutant had about half the Vmax of the wild-type and a 12-fold higher Km for PPi, whereas NIN and LL mutations lowered the Vmax by 12- and 2-fold, respectively, with relatively small effects on substrate Km values (the NNN mutant was insoluble/inactive). The NIN mutation resulted in a low-activity oligomerized enzyme form, with very little monomer formation. Activity staining on native PAGE gels as well as gel-filtration studies demonstrated that the monomer was the sole enzymically active form. Possible implications of the oligomerization status of UGPase for post-translational regulation of the enzyme are discussed.
Deficiency of dolichyl-P-Man:Man7GlcNAc2-PP-dolichyl mannosyltransferase causes congenital disorder of glycosylation type Ig
Authors: THIEL Christian , SCHWARZ Markus , HASILIK Martin , GRIEBEN Ulrike , HANEFELD Folker , LEHLE Ludwig , FIGURA Kurt von , KÖRNER Christian
Published: September 2002

Deficiency of the endoplasmic reticulum enzyme dolichyl-phosphate mannose (Dol-P-Man):Man7GlcNAc2-PP-dolichyl mannosyltransferase leads to a new type of congenital disorder of glycosylation, designated type Ig. The patient 1 presented with a multisystemic disorder with microcephaly, developmental retardation, convulsions and dysmorphic signs. The isoelectric focusing pattern of the patient's serum transferrin showed the partial loss of complete N-glycan side chains. In skin fibroblasts from the patient, the activity of Dol-P-Man:Man7GlcNAc2-PP-Dol mannosyltransferase was severely reduced leading to the accumulation of Man7GlcNAc2-PP-Dol, which was transferred to newly synthesized glycoproteins. Sequencing of the Dol-P-Man:Man7GlcNAc2-PP-Dol mannosyltransferase cDNA revealed a compound heterozygosity for two point mutations, leading to the exchange of leucine158 for a proline residue and a premature translation stop with loss of the C-terminal 74 amino acids. The parents were heterozygous for one of the two mutations. Retroviral expression of the wild-type Dol-P-Man:Man7GlcNAc2-PP-Dol mannosyltransferase cDNA in patient's fibroblasts normalized the mannosyltransferase activity.
Biphasic translocation of Bax to mitochondria
Authors: CAPANO Michela , CROMPTON Martin
Published: September 2002

Using green fluorescent protein-tagged Bax, we demonstrate that Bax is sequestered from the cytosol of cardiomyocytes in two distinct phases following the induction of apoptosis with staurosporine. In the first phase, lasting several hours, Bax removal from the cytosol was relatively small. In the second phase, Bax was very largely removed from the cytosol and sequestered into large aggregates associated with the mitochondria. To test which of the phases involved cytochrome c release, cells were transfected with a red fluorescent protein—cytochrome c fusion. The cytochrome c fusion protein was accumulated by mitochondria of healthy cells and was released by staurosporine in phase 1. When green fluorescent protein—Bax was immunoprecipitated from extracts of cells in phase 1 and phase 2, the voltage-dependent anion channel (mitochondrial outer membrane) and the adenine nucleotide translocase (mitochondrial inner membrane) were also precipitated. These data support a two-phase model of Bax translocation in which Bax targets the mitochondrial intermembrane contact sites and releases cytochrome c in the first phase, and is then packaged into large aggregates on mitochondria in the second.
The metalloendopeptidase nardilysin (NRDc) is potently inhibited by heparin-binding epidermal growth factor-like growth factor (HB-EGF)
Authors: HOSPITAL Véronique , NISHI Eiichiro , KLAGSBRUN Michael , COHEN Paul , SEIDAH Nabil G. , PRAT Annik
Published: September 2002

Nardilysin (N-arginine dibasic convertase, or NRDc) is a cytosolic and cell-surface metalloendopeptidase that, in vitro, cleaves substrates upstream of Arg or Lys in basic pairs. NRDc differs from most of the other members of the M16 family of metalloendopeptidases by a 90 amino acid acidic domain (DAC) inserted close to its active site. At the cell surface, NRDc binds heparin-binding epidermal growth factor-like growth factor (HB-EGF) and enhances HB-EGF-induced cell migration. An active-site mutant of NRDc fulfills this function as well as wild-type NRDc, indicating that the enzyme activity is not required for this process. We now demonstrate that NRDc starts at Met49. Furthermore, we show that HB-EGF not only binds to NRDc but also potently inhibits its enzymic activity. NRDc—HB-EGF interaction involves the 21 amino acid heparin-binding domain (P21) of the growth factor, the DAC of NRDc and most probably its active site. Only disulphide-bonded P21 dimers are inhibitory. We also show that Ca2+, via the DAC, regulates both NRDc activity and HB-EGF binding. We conclude that the DAC is thus a key regulatory element for the two distinct functions that NRDc fulfills, i.e. as an HB-EGF modulator and a peptidase.
A novel Rho GTPase-activating-protein interacts with Gem, a member of the Ras superfamily of GTPases
Authors: ARESTA Sandra , TAND-HEIM Marie-France de , BÉRANGER Florence , GUNZBURG Jean de
Published: September 2002

Gem is a Ras-related protein whose expression is induced in several cell types upon activation by extracellular stimuli. With the aim of isolating the cellular partners of Gem that mediate its biological activity we performed a yeast two-hybrid screen and identified a novel protein of 970 amino acids, Gmip, that interacts with Gem through its N-terminal half, and presents a cysteine-rich domain followed by a Rho GTPase-activating protein (RhoGAP) domain in its C-terminal half. The RhoGAP domain of Gmip stimulates in vitro the GTPase activity of RhoA, but is inactive towards other Rho family proteins such as Rac1 and Cdc42; it is also specific for RhoA in vivo. The same is true for the full-length protein, which is furthermore able to down-regulate RhoA-dependent stress fibres in Ref-52 rat fibroblasts. These findings suggest that the signalling pathways controlled by two proteins of the Ras superfamily, RhoA and Gem, are linked via the action of the RhoGAP protein Gmip (Gem-interacting protein).
Substrate specificity of the metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) assessed by mutagenesis and analysis of synthetic peptides: substrate residues distant from the scissile bond are critical for proteolysis
Authors: LAURSEN Lisbeth S. , OVERGAARD Michael T. , NIELSEN Claus G. , BOLDT Henning B. , HOPMANN Kathrin H. , CONOVER Cheryl A. , SOTTRUP-JENSEN Lars , GIUDICE Linda C. , OXVIG Claus
Published: September 2002

Human pregnancy-associated plasma protein-A (PAPP-A) cleaves insulin-like growth factor (IGF) binding protein-4 (IGFBP-4), causing a dramatic reduction in its affinity for IGF-I and -II. Through this mechanism, PAPP-A is a regulator of IGF bioactivity in several systems, including the human ovary and the cardiovascular system. PAPP-A belongs to the metzincin superfamily of zinc metalloproteinases, and is the founding member of a fifth metzincin family, the pappalysins. Herein, we first determined that PAPP-A cleaves IGFBP-4 at a single site (Met-135/Lys-136), and we analysed the influence of ionic strength, pH and zinc ion concentration on the cleavage reaction. Secondly, we sought to delineate the role of substrate residues in PAPP-A-mediated cleavage by the construction and analysis of 30 IGFBP-4 mutants in which various residues were replaced by alanine, by the analysis of eight mutants of IGFBP-5 (found recently to be a second PAPP-A substrate), and by cleavage analysis of synthetic peptides derived from IGFBP-4. Our data reveal a complex mode of substrate recognition and/or binding, pointing at important roles for several basic residues located up to 16 residues N-terminal to the scissile bond. An unexpected parallel can be drawn with an intracellular enzyme, the mitochondrial processing peptidase, that may help us to understand properties of the pappalysins. Further, proteinase-resistant variants of IGFBP-4 and −5, presented here, will be useful tools for the study of proteolysis in cell-based systems, and our finding that a synthetic peptide can be cleaved by PAPP-A provides the basis for development of quantitative assays for the investigation of PAPP-A enzyme kinetics.
Transport of a neurotoxicant by molecular mimicry: the methylmercury–l-cysteine complex is a substrate for human L-type large neutral amino acid transporter (LAT) 1 and LAT2
Authors: SIMMONS-WILLIS Tracey A. , KOH Albert S. , CLARKSON Thomas W. , BALLATORI Nazzareno
Published: September 2002

Methylmercury (MeHg) readily crosses cell membrane barriers to reach its target tissue, the brain. Although it is generally assumed that this rapid transport is due to simple diffusion, recent studies have demonstrated that MeHg is transported as a hydrophilic complex, and possibly as an l-cysteine complex on the ubiquitous L-type large neutral amino acid transporters (LATs). To test this hypothesis, studies were carried out in Xenopus laevis oocytes expressing two of the major L-type carriers in humans, LAT1—4F2 heavy chain (4F2hc) and LAT2—4F2hc. Oocytes expressing LAT1—4F2hc or LAT2—4F2hc demonstrated enhanced uptake of [14C]MeHg when administered as the l-cysteine or d,l-homocysteine complexes, but not when administered as the d-cysteine, N-acetyl-l-cysteine, penicillamine or GSH complexes. Kinetic analysis of transport indicated that the apparent affinities (Km) of MeHg—l-cysteine uptake by LAT1 and LAT2 (98±8 and 64±8μM respectively) were comparable with those for methionine (99±9 and 161±11μM), whereas the Vmax values were higher for MeHg—l-cysteine, indicating that it may be a better substrate than the endogenous amino acid. Uptake and efflux of [3H]methionine and [14C]MeHg—l-cysteine were trans-stimulated by leucine and phenylalanine, but not by glutamate, indicating that MeHg—l-cysteine is both a cis- and trans-substrate. In addition, [3H]methionine efflux was trans-stimulated by leucine and phenylalanine even in the presence of an inwardly directed methionine gradient, demonstrating concentrative transport by both LAT1 and LAT2. The present results describe a major molecular mechanism by which MeHg is transported across cell membranes and indicate that metal complexes may form a novel class of substrates for amino acid carriers. These transport proteins may therefore participate in metal ion homoeostasis and toxicity.
Inhibitor specificity of recombinant and endogenous caspase-9
Authors: RYAN Ciara A. , STENNICKE Henning R. , NAVA Victor E. , BURCH Jennifer B. , HARDWICK J. Marie , SALVESEN Guy S.
Published: September 2002

Apoptosis triggered through the intrinsic pathway by radiation and anti-neoplastic drugs is initiated by the activation of caspase-9. To elucidate control mechanisms in this pathway we used a range of synthetic and natural reagents. The inhibitory potency of acetyl-Asp-Glu-Val-Asp-aldehyde ('Ac-DEVD-CHO'), benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone ('Z-VAD-FMK') and the endogenous caspase inhibitor X-chromosome-linked inhibitor of apoptosis protein ('XIAP') against recombinant caspase-9 were predictive of the efficacy of these compounds in a cell-free system. However, the viral proteins CrmA and p35, although potent inhibitors of recombinant caspase-9, had almost no ability to block caspase-9 in this system. These findings were also mirrored in cell expression studies. We hypothesize that the viral inhibitors CrmA and p35 are excluded from reacting productively with the natural form of active caspase-9 in vivo, making the potency of inhibitors highly context-dependent. This is supported by survival data from a mouse model of apoptosis driven by Sindbis virus expressing either p35 or a catalytic mutant of caspase-9. These results consolidate previous findings that CrmA is a potent inhibitor of caspase-9 in vitro, yet fails to block caspase-9-mediated cell death.
A catalytic consensus motif for d-mannitol 2-dehydrogenase, a member of a polyol-specific long-chain dehydrogenase family, revealed by kinetic characterization of site-directed mutants of the enzyme from Pseudomonas fluorescens
Authors: KLIMACEK Mario , NIDETZKY Bernd
Published: September 2002

Lys-295, Asn-300 and His-303 of d-mannitol 2-dehydrogenase from Pseudomonas fluorescens were mutated individually into alanine (K295A, N300A and H303A respectively). Purified mutants displayed catalytic efficiencies for NAD+-dependent oxidation of d-mannitol 300-fold (H303A), 1000-fold (N300A) and approx. 400000-fold (K295A) below the wild-type level. Comparison of primary kinetic isotope effects on kinetic parameters for d-fructose reduction by wild-type and mutants at pH10.0 demonstrate that Asn-300 has an auxiliary role in stabilization of the transition state of hydride transfer, and His-303 contributes to substrate positioning. The large solvent isotope effect of 11±1 on kcat for mannitol oxidation by K295A at pH(2H) 10.5 suggests a role for Lys-295 in general base enzymic catalysis. Positional conservation of Lys-295, Asn-300 and His-303 across a family of polyol-specific long-chain dehydrogenases suggests a unique catalytic signature: Lys-Xaa4-Asn-Xaa2-His (where ‘Xaa’ denotes ‘any amino acid').
Grain quality of flooded rice is affected by season, nitrogen rate, and plant type
Authors: Borrell A. K. , Garside A. L. , Fukai S. , Reid D. J.
Published: September 2002

Quality of grain, next to yield, is the most important factor for rice
(Oryza sativa L.) production in semi-arid tropical
Australia. Studies were undertaken in the Burdekin River Irrigation Area of
northern Australia to improve rice grain quality through nitrogen
fertilisation. This paper reports the results of 4 experiments comparing the
response of 3 rice genotypes differing in maturity and stature to 5 rates of
applied nitrogen (0, 70, 140, 210, and 280 kg/ha) over 4 seasons (2 wet
and 2 dry seasons). The components of grain quality studied were endosperm
chalkiness, whole grain millout, grain size, alkali digestion (gelatinisation
temperature), and grain protein. This paper also examines the suitability of
the 3 genotypes as parental material in breeding programs aimed at selecting
for specific grain quality attributes.
Starbonnet was identified as a potential parent in breeding programs which aim
to specifically select for reduced chalkiness and high millout in low N
environments. Selection for lower chalkiness, and higher millout and protein
concentration, should occur in a wide range of target environments to account
for the seasonal variation observed in these parameters. Grain size appeared
to be affected more by genetic than agronomic factors, since grain length and
breadth were largely unaffected by N rate, yet genotypic differences were
found for both parameters in all experiments. Newbonnet grain was long and
slender, suggesting this genotype would be a suitable parent in breeding
programs aimed at improving grain appearance. The response of alkali digestion
to N rate and genotype was small for all seasons.
The importance of developing N fertiliser strategies that optimise both grain
yield and quality was highlighted by differences in the responses of grain
protein and grain yield to N rate. A number of linkages were examined among
various components of grain quality. However, the magnitude of these linkages
was small, suggesting that selection for one quality component should not be
at the expense of selection for another.
Hardseededness and the pattern of softening in Biserrula pelecinus L., Ornithopus compressus L., and Trifolium subterraneum L. seeds
Authors: Loi A. , Cocks P. S. , Howieson J. G. , Carr S. J.
Published: September 2002

Experiments measuring seed bank size, hardseededness, and seed softening of
biserrula (Biserrula pelecinus L.) were conducted at
Merredin and Perth in Western Australia. At Merredin, a mixture of 2 biserrula
accessions was grazed, shallow cultivated, or left uncultivated and ungrazed.
Seed bank size, seedling regeneration, and seed softening were measured over 2
years. At Perth, softening of biserrula, yellow serradella, and subterranean
clover seeds grown at 2 sites (Binnu and Northam) was compared on the soil
surface and after burial at 2 and 10 cm over a period of 2 years.
Seed bank size of biserrula at Merredin ranged from 14000 to 17500
seeds/m2. Regeneration was greater in the second
year (800–1700 seedlings/m2) than in the first
year (40–600 seedlings/m2). In both years the
shallow cultivated treatment recorded the highest number of seedlings.
About 90% of biserrula and serradella seed remained hard after 2 years
on the soil surface, compared with only about 10% of subterranean
clover. Serradella softened more rapidly when buried 2 cm below the soil
surface (8–12% hard) than it did on the soil surface
(84–92% hard) (P<0.05). In contrast, the
softening of subterranean clover decreased with increasing depth. Biserrula
was intermediate, although it too softened most rapidly at 2 cm
(78–95% hard compared with 82–97% on the surface)
(P<0.05). Rate of seed softening in all species
decreased with increasing depth of burial below 2 cm. Of the 4 accessions of
biserrula, an accession from Greece (83% hard after 2 years exposure)
was significantly softer than the other accessions.
The results indicate that biserrula is very hardseeded, although there is
sufficient variation in hardseededness for the selection of somewhat softer
lines. Its pattern of softening suggests that biserrula may be successful in
the ley farming system (crop/pasture rotations) of southern Australia.
Diallel analysis of vernalisation responses in spring rape (Brassica napus L.): a basis for adaptation to a Mediterranean environment
Authors: Dahanayake S. R. , Galwey N. W.
Published: September 2002

The capacity for vernalisation varies among genotypes of spring rape,
Brassica napus L., and any vernalisation response
reduces their suitability for cultivation in the Mediterranean environment of
south-western Western Australia. The genetic basis of this variation was
studied in the F1 generation of a diallel cross among 5
inbred genotypes derived from a cross between 2 cultivars differing in
sensitivity to vernalisation. In non-vernalised plants the number of leaf
nodes at flowering, time to flowering, stem height at flowering, and time to
initiation of stem elongation showed substantial genetic variation, both
additive and dominance effects being significant. The time to initiation of
stem elongation was not vernalisation-sensitive, but for the other 3
characters the response to vernalisation showed significant genetic variation,
largely additive. The genetic effects controlling the number of leaf nodes at
flowering were clearly different from those controlling stem height at
flowering, and hence could be manipulated independently by selection.
Moreover, the genetic effects controlling these characters in the absence of
vernalisation were different from those controlling the response to
vernalisation. Discontinuities in the distributions of the parent means and
other statistics for the number of leaf nodes at flowering suggested that
major Mendelian genes may largely control this character and its response to
vernalisation. Comparison of the number of leaf nodes at flowering in the
presence and absence of a vernalising stimulus is therefore suggested as a
selection criterion for the elimination of vernalisation response. However, in
the absence of a vernalising stimulus, the time to flowering will be the
character easiest to influence by selection.
Prior crop and residue incorporation time affect the response of paddy rice to fertiliser nitrogen
Authors: Ockerby S. E. , Garside A. L. , Holden P. D. , Adkins S. W.
Published: September 2002

Crop residues are an important source of nitrogen (N) for rice
(Oryza sativa L.). The objective of this research was to
determine how the supply of mineral N from different prior crops or fallow
might affect the growth and yield of rice. The study also tested whether N use
by rice might be improved by timing the application of inorganic fertiliser N
to supplement the N mineralised after prior crops. Experiments consisted of
fallow, or cereal or legume crops in the dry-season followed by wet-season
rice; and fallow, or cereal or legume crops in the wet- season followed by
dry-season rice. Urea at one-third of the rate required for optimum rice yield
was applied at 3 times during the rice crop: sowing, permanent flood,
and/or panicle initiation. The prior fallow and crop treatments
significantly influenced the growth and yield of rice crops. After a fallow,
the pattern of soil N mineralisation promoted vegetative growth but was
limiting during grain-filling. In contrast, after a cereal crop, rice
vegetative growth was limited but grain-filling was promoted. Legume prior
crops promoted both vegetative and grain growth. The benefits derived from
growing the cereal or legume crops before rice, in terms of replacing
fertiliser N, were dependent on the time at which fertiliser N was applied to
the rice crop. In particular, legume crops frequently nullified the rice
growth responses to fertiliser N. The results demonstrated that fallow and
prior crops can alter the amount and timing of mineral N supply to a rice
crop. Farmers should consider including a legume crop in rotation with rice
because legumes supply N, which increases rice yield and reduces the
requirement for fertiliser N. Cereal crops also contribute N, although farmers
who use a cereal rotation should monitor the soil and crop N status during
early rice growth, and supply extra fertiliser N to alleviate N deficiency.
The uptake and use of nitrogen by paddy rice in fallow, cereal, and legume cropping systems
Authors: Ockerby S. E. , Garside A. L. , Adkins S. W.
Published: September 2002

In a previous paper, we reported that prior crops either increased or
decreased the yield of paddy rice (Oryza sativa L.) and
altered its response to fertiliser N. We considered that rice yield responses
to prior crop might have reflected the uptake of crop residue N and the
efficiency of its use to produce grain. Experiments consisted of dry-season
grain or legume crops, or fallow, followed by wet-season rice (cv. Lemont);
and wet-season grain or legume crops, or fallow, followed by dry-season rice.
Urea at one-third of the rate required for optimum rice yield was applied at 3
stages of rice crop growth: sowing, permanent flood, and/or panicle
initiation. Soil N supplied 4.1 to 6.5 g N/m2 to the
rice crop, depending on the season. Rice also recovered 0 to 0.25 of the N in
the residue of a prior maize crop and 0.23 to 0.57 of the N in grain legume
residues or a legume green manure crop; the fraction was greater if fertiliser
N was not applied. Increased N uptake was the major contributor to heavier
yield. The relationship between grain yield and crop N content was mostly
linear, and thus physiological efficiency of N use for rice grain production
was essentially constant across the range of environments provided by
fertiliser N and cropping system treatments in this study. In experiments
where fertiliser N was applied, there were small effects of prior cereal and
legume cropping treatments on physiological efficiency. In contrast, without
fertiliser N application, physiological efficiency was increased by prior
cereal and legume crops, which likely resulted from a greater congruence
between the N demand of the rice crop, and the N supply from the soil and
incorporated residue, when compared with a fallow treatment.
The Agouti gene: a positional candidate for recessive self-colour pigmentation in Australian Merino sheep
Authors: Parsons Y. M. , Fleet M. R. , Cooper D. W.
Published: September 2002

The occurrence of self-colour pigmentation in the Australian Merino wool flock
is of considerable economic importance. The Agouti gene
is believed to be responsible for the recessive expression of pigmented
fleece. Using comparative mapping information we have investigated the
putative homologous ovine map positon of the Agouti gene
for linkage to the recessive self-colour phenotype of Australian Merino sheep.
Significant results were observed with microsatellites previously mapped to
ovine chromosome 13. Comparative data suggest that the ovine
Agouti gene would map to the same chromosome, making the
Agouti gene a positional candidate for the self-colour phenotype.
Legume and opportunity cropping systems in central Queensland. 1. Legume growth, nitrogen fixation, and water use
Authors: Armstrong R. D. , McCosker K. , Johnson S. B. , Millar G. , Walsh K. B. , Kuskopf B. , Probert M. E. , Standley J.
Published: September 2002

An experiment, established on a cracking clay (Vertisol) at Emerald, central
Queensland, studied the dry matter (DM) production, nitrogen (N) fixation, and
water use of several potential ley-legume species over 4 seasons
(1994–1997). Four ley legumes (siratro,
Macroptilium atropurpureum cv. Siratro; lucerne,
Medicago sativa cv. Trifecta; lablab,
Lablab purpureus cv. Highworth; and desmanthus,
Desmanthus virgatus cv. Marc) were compared with a pulse
(mungbean, Vigna radiata cv. Satin), and grain sorghum
(Sorghum bicolor) was included as a non-legume control.
Overall, the annual legumes lablab (17.5 t/ha) and mungbean (13.4
t/ha) and the perennial siratro (16.2 t/ha) accumulated more DM than
the perennials lucerne (9.6 t/ha) and desmanthus (7.1 t/ha). Lucerne
produced little DM in its first year, but in later years had similar
production to siratro and lablab. Desmanthus produced >4 t/ha of DM in
the first year but barely survived during later seasons.
Annual legumes grew faster and exhausted soil water more rapidly than the
perennials. The perennials were able to extract more water from the soil than
the annual legumes and sorghum, but were inefficient at converting small to
moderate rainfall events (25–50 mm) into DM production. During the
fallow following the growth of lablab and mungbean, nitrate-N in soil
increased and was always greater at the time of re-sowing than for the
perennial legumes and sorghum.
Initially, the 2 annual legumes derived a high proportion (50% to
>70%) of their above-ground N from fixation (%Ndfa) but this
declined as the experiment progressed to low values (<13%) in the
third and fourth years, reflecting increased supply of nitrate from the soil.
In contrast, %Ndfa peaked at 72% for siratro and >90%
for lucerne, and remained high (25–50%) throughout the
experiment. N fixation rates were strongly negatively correlated with soil
nitrate. Over the 4 years, siratro fixed 161 kg N/ha, lucerne 120, lablab
119, mungbean 78, and desmanthus 19 based on above-ground biomass. Mungbean
had a net negative N balance (–80 kg N/ha) due to N exported in
grain.
Legume and opportunity cropping systems in central Queensland. 2. Effect of legumes on following crops
Authors: Armstrong R. D. , McCosker K. , Walsh K. , Millar G. , Kuskopf B. , Probert M. E. , Johnson S. , Standley J.
Published: September 2002

Poor yields and low grain protein in cereal crops resulting from declining
soil fertility, especially nitrogen (N), are major threats to the grains
industry in central Queensland. The effect of 4 different pasture-ley legumes
[siratro (Macroptilium atropurpureum cv. Siratro),
lucerne (Medicago sativa cv. Trifecta), lablab
(Lablab purpureus cv. Highworth), and desmanthus
(Desmanthus virgatus cv. Marc)] on grain yield and
quality of sorghum crops was compared with that of a pulse (mungbean;
Vigna radiata cv. Satin) or continuous cropping with
grain sorghum (Sorghum bicolor).
Legume leys consistently resulted in large increases in grain yield
(188–272%), N uptake by sorghum (145–345%), and
grain protein (0.21–7.0% increase in grain protein) in sorghum
test-crops compared with continuous sorghum crops to which no fertiliser N had
been added. The positive effect of legumes persisted up to 3 sorghum
test-crops after only 1 year of legumes, although by the third year the effect
was comparatively small. Mungbean and lablab generally produced the largest
benefit in sorghum test-crops in the first year after legumes, whereas
desmanthus and lucerne produced the least benefit. Adding fertiliser N (up to
75 kg N/ha) significantly improved grain yields and N uptake of sorghum
test-crops in 3 of 4 years. However, responses to fertilisers were less than
those resulting from legumes, which was ascribed to increased availability of
legume N to sorghum.
Legumes progressively increased soil nitrate in all subsequent sorghum
test-crops (compared with continuous sorghum crops), rising from
6.8–18.9 kg NO3-N/ha after 1 year of legumes
to 24.2–59.6 kg NO3-N/ha after 3 years of
legumes. There was little difference between the legumes in their ability to
increase soil nitrate, with the exception of desmanthus, which consistently
resulted in the lowest amount of soil nitrate for subsequent test-crops and
lowest uptake of N by these crops.
Plant-available water content (PAWC) at planting of the sorghum test-crop was
only significantly (P<0.05) affected by previous
species in 1997, when it was lowest in plots previously sown to siratro and
lucerne and highest in sorghum and mungbean plots. In both 1996 and 1997,
plots sown to sorghum had significantly higher PAWC at anthesis and grain
maturity when previous plots were sorghum rather than legumes.
Nitrogen and sulfur dynamics of contrasting grazed pastures
Authors: Chen Wen , Blair Graeme , Scott Jim , Lefroy Rod
Published: September 2002

The experimental area was located at the Big Ridge 2 site, CSIRO, Chiswick
(30°31′S, 151°39′E), 20 km south of Armidale, New South
Wales, Australia. The site was established in 1955. In March 1966, phalaris
and white clover were sown and pastures were fertilised annually with
superphosphate until 1993. There were 3 pasture treatments, each with 2
replicates: degraded pasture (low phalaris content), phalaris dominant, and
phalaris–white clover. Each of 6 experimental plots was divided into 3
strata. Two representative areas 1 m by 0.5 m were selected in each stratum of
each treatment. The selected areas were labelled with 34S-enriched (90%) elemental sulfur and 15N-enriched (99%) NH4Cl solution. All plots were
grazed continuously by sheep.
No effect of pasture type on N leaching was apparent in this experiment.
Seasonal variation of total soil mineral N in different soil layers, low
15N recovery down to 60 cm soil depth, and low
nitrate-N concentrations in drainage water obtained in this experiment suggest
that synchronisation of pasture growth with mineralisation and nitrification,
together with ammonium domination of the soil N system, is the key ecological
feature in preventing N leaching in this environment.
Unlike N, potential S leaching was found with evidence of a large amount of
sulfate stored deeper in the soil profile and high S concentrations in
drainage water. High KCl-40 extractable S concentration in the top 20 cm soil
layers was associated with the long history of superphosphate application.
Long-term applications of superphosphate (1967–93), together with an
increase in sulfate sorption capacity at lower soil depths, resulted in a
large amount of sulfate stored at greater depth. However, retention of the
34S applied in 1995 in the top 10 cm soils suggests
that sulfate-S movement down the soil profile is slow.
Water use efficiency and water use of Mediterranean annual pastures in southern Australia
Authors: Bolger T. P. , Turner N. C.
Published: September 2002

There is a perception in the farming and research communities that annual
pastures have low produc- tivity and water use, and contribute
disproportionately to problems of rising watertables and dryland salinity. Our
aim was to determine potential pasture production in relation to water use and
the influence of management factors on this relationship. Experiments were
initiated at 4 locations along a gradient of 300–1100 mm annual rainfall
across the Western Australian agricultural zone. At each site a high input
treatment was compared with a low input control. There was a strong linear
relationship between water use and pasture production up to 440 mm of growing-
season water use. After 30 mm of water use the potential pasture production
was 30 kg/ha.mm. An upper limit to pasture production may be reached at
about 12 000 kg/ha in this environment due to rainfall distribution
patterns and soil water holding capacity in the root-zone. Although pasture
production was increased by as much as 3500 kg/ha, water use was generally
similar or only slightly more for high input compared with control plots. The
marginally higher water use by the high input pastures resulted in an extra 18
mm of water extracted from the subsoil at one location by the end of the third
season. A drier subsoil may provide a buffer for storing excess rainfall and
reduce deep drainage. Estimated drainage was small at low rainfall sites so
even marginal increases in water use by highly productive annual pastures
could play a significant role in reducing water loss to deep drainage and
mitigating water-table rise and secondary salinisation in low rainfall
regions. Management practices aimed at promoting early growth and adequate
leaf area should maximise water use, water use efficiency, and yield. The
linear relationship defining potential pasture production provides a useful
benchmark to farmers.
Effect of copper fungicide on Colletotrichum gloeosporioides and other microorganisms on avocado leaves and fruit
Authors: Stirling A. M. , Stirling G. R. , Pegg K. G. , Hayward A. C.
Published: September 2002

An investigation into the effect of copper fungicide on non-target
microorganisms on avocado (cvv. Hass and Nabal) showed that populations of
filamentous fungi, yeasts, and bacteria on leaves were at least 10-fold less
after a single application of copper than on unsprayed leaves. This
detrimental effect of copper on microorganisms on the phylloplane was
confirmed in random samples collected from 5 commercial avocado orchards with
regular pesticide spray programs but where isolated unsprayed trees were also
available. An intensive 16-month study was carried out on 2 adjacent orchards,
1 that was sprayed with copper fungicide at monthly intervals from October to
April each year, and the other that had not been sprayed for at least 6 years.
Populations of filamentous fungi, yeasts, and bacteria on leaves and fruit
were 10–100-fold lower in the sprayed orchard than in the unsprayed
orchard. However, populations in the sprayed orchard recovered during the
winter non-spray periods to levels comparable to the unsprayed orchard. In
1993 and 1994, fruit were harvested from both orchards, ripened, and assessed
for anthracnose caused by
Colletotrichum gloeosporioides. In both years, there was
significantly less disease in unsprayed fruit (mean disease rating 1.13 and
0.32, respectively) than in sprayed fruit (mean disease rating 1.83 and 2.18,
respectively). These results show that copper fungicide is detrimental to
phylloplane microorganisms and suggest that those organisms are providing some
natural suppression of C. gloeosporioides on avocado.