Tolerance of wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) to high soil and solution selenium levels

The fertilisation of wheat crops with Se is a cost-effective method of enhancing the concentration of organic Se in grain, in order to increase the Se intake of animals and humans. It is important to avoid phytotoxicity due to over-application of Se. Studies of phytotoxicity of Se in wheat grown in Australia, where rainfall and grain yield are usually relatively low, have not been reported previously, and overseas studies have had varied results. This study used trials conducted in the field, glasshouse and laboratory to assess Se phytotoxicity in wheat. In field trials that used rates of up to 120 g ha^–1Se as selenate, and in pilot trials that used up to 500 g ha^–1 Se soil-applied or up to 330 g ha^–1 Se foliar-applied, with soils of low S concentrations (2–5 mg kg^–1), no Se toxicity symptoms were observed. In pot trials of four weeks duration, the critical tissue level for Se toxicity was around 325 mg kg^–1 DW, a level attained by addition to the growth medium of 2.6 mg kg^–1 Se as selenate. Solution concentrations above 10 mg L^–1 Se inhibited early root growth of wheat in laboratory studies, with greater inhibition by selenite than selenate. For selenite, Se concentrations around 70 mg L^–1 were required to inhibit germination, while for selenate germination % was unaffected by a solution concentration of 150 mg L^–1 Se. Leaf S concentration and content of wheat increased three-fold with the addition of 1 mg kg^–1 Se as selenate to the growth medium. This effect is probably due to the induction of the S deficiency response of the main sulphate transporter. This study found wheat to be more Se-tolerant than did earlier studies of tobacco, soybeans and rice. We conclude that Se phytotoxicity in wheat will not be observed at the range of Se application rates that would be used to increase grain Se for human consumption (4–200 g ha^–1 Se as selenate, which would result in soil and tissue levels well below those seen in the above studies), even when – as is common in Australia – soil S concentration and grain yield are low.     

Purification of theN-acetylglucosaminide α(1–3/4) fucosyltransferase of human milk

TheN-acetylglucosaminide (1–3/4)fucosyltransferase has been purified 1.8×10⁶-fold from human milk by ion-exchange chromatography, affinity chromatography of GDP-agarose and HPLC. The (1–3/4)fucosyltransferase behaves in gel filtration-HPLC as a molecule of M_r 98 000, and differs from the (1–3)fucosyltransferase which behaves like a molecule of about M_r 47 000. The enzyme is a glycoprotein, and the purified preparation appears in SDS polyacrylamide gel electrophoresis as a band of M_r 44 000. The results present the first purification of human milk (1–3/4)fucosyltransferase to apparent homogeneity, and suggest that the (1–3/4)- and (1–3)fucosyltransferases of human milk differ in their native molecular sizes, the former being a dimer of two subunits.     

Enhancement of production of cloned α-amylase by lactic acid feeding from recombinant Saccharomyces cerevisiae using a SUC2 promoter

-Amylase production was higher (13 units ml^–1) when a recombinant Saccharomyces cerevisiae containing a SUC2 promoter was grown with 10 g lactic acid l^–1 than without addition (8 units ml^–1). With continuous lactic acid feeding in the inducing phase, -amylase increased to 79 units ml^–1 in a 1-l jar fermenter.     

O-Glycosidically linked oligosaccharides from peptidorhamnomannans ofSporothrix schenckii

-Elimination of peptidorhamnomannans purified from yeast-like and mycelial phases ofSporothrix schenckii released neutral and acidic reduced oligosaccharides that were O linked to serine and/or threonine. Man-(1–2)Man-ol, Rha(1–3)Man(1–2)Man-ol, Rha(1–4)GlcA(1–2)Man(1–2)Man-ol, and Rha(1–4)[Rha(1–2)] GlcA(1–2)Man(1–2)Man-ol were characterized based on methylation analysis, proton magnetic resonance and fast atom bombardment mass spectrometry.Abbreviations FAB fast atom bombardment - GLC gas liquid chromatography - GlcA d-glucopyranosyluronic acid - Man d-mannopyranose - Man-ol d-mannitol - MS mass spectrometry - NMR nuclear magnetic resonance - Rha l-rhamnopyranose     

TNF/LTα double knockout mice display abnormal inflammatory and regenerative responses to acute and chronic liver injury

Following acute liver injury, hepatocytes divide to facilitate regeneration. However, during chronic injury, hepatocyte proliferation is typically blocked and repair is mediated through liver progenitor (oval) cells. Signalling of the p55 tumour necrosis factor (TNF) receptor is central to these processes. Two ligands for p55 are known: TNF and lymphotoxin-alpha (LT). However, one study suggests that another exists that mediates liver injury following viral challenge. We have therefore investigated whether ligands other than TNF and LT are required for liver regeneration following either acute or chronic injury. Wild-type and double TNF/LT knockout (TNF^–/–LT^–/–) mice were subjected to either partial hepatectomy (PHx) or a choline-deficient ethionine-supplemented (CDE) diet. Proliferating hepatocytes, oval cells and inflammatory cells were identified and quantified in liver sections by immunohistochemistry. Liver inflammatory cells were characterised by cell surface antigen expression. Liver damage and mortality were monitored. Both hepatocyte and oval cell proliferation was reduced in TNF^–/–LT^–/– mice. Lymphocyte clusters were evident in all TNF^–/–LT^–/– livers and were heterogeneous, comprising B and T lymphocytes. PHx evoked liver inflammation in TNF^–/–LT^–/– but not wild-type mice, whereas no difference was apparent between genotypes in CDE experiments. Thus, TNF/LT signalling mediates liver regeneration involving both hepatocytes and progenitor cells. The hyper-inflammatory response following PHx in TNF^–/–LT^–/– animals, which is absent following CDE-induced injury, demonstrates that the two forms of liver injury evoke discrete inflammatory responses and provides a model in which such differences can be examined further.     

Changing Global Climate: Historical Carbon and Nitrogen Budgets and Projected Responses of Ohio’s Cropland Ecosystems

As the evidence of global climate change continues to mount, its consequences for cropland productivity assume particular significance. Against the backdrop of past agricultural practices, simulation models offer a glimpse into the future, showing the effect of temperature changes on crop production. In this study, we first quantified the carbon (C) and nitrogen (N) budgets of Ohios cropland ecosystems using inventory yield data of corn for grain, oat, and all wheat for the period 1866–1996 and soybean for the period 1924–96. Then we explored the responses of Ohios continuous soybean croplands to changes in temperature, carbon dioxide (CO₂) concentration, initial soil organic C and N (SOC-N) pools, soil texture, and management practices by developing a simple cropland ecosystem model (CEM) and performing a long-term sensitivity analysis. Finally, CEM simulations were evaluated against independent observations of SOC values (0–19 cm) averaged over 470 northwest Ohio sites between 1954 and 1987 under conventional tillage and rotations of corn–soybean–winter wheat by using the historical yield data (r ² = 0.8). The C contents per hectare of crop harvests increased by 178% for oats, 300% for corn for grain, and 652% for all wheat between 1866 and 1996 and by 305% for soybean between 1924 and 1996. Ohio croplands acted as C–N sources, releasing average net ecosystem emissions (NEE), including the removal of harvested C–N, of 4,598 kg CO₂ ha^–1 and 141 kg N ha^–1 in 1886 and 205 kg CO₂ ha^–1 (except for the corn-for-grain cropland) and 39 kg N ha^–1 in 1996. The continuous corn croplands continued to become a C sink, sequestering 255 kg C ha^–1 in 1996. Results of the sensitivity analysis for Ohios continuous soybean croplands revealed that the SOC pool increased by 6.9% and decreased by 7.5% in response to a doubled CO₂ concentration and a temperature increase of 2.8°C over 100 years, respectively. The sequestration potential of the SOC pool increased by 6.5% at a rate of 24.6 kg C ha^–1 y^–1 for the same period with finer soil texture (loam to silty clay loam). The shift from conventional to conservation residue practice led to an 11% increase in the steady-state SOC storage at a rate of 42 kg C ha^–1 y^–1 for 100 years.     

Isolation and characterization of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by the mating hormone a-factor

Summary Nine independent mutants which are supersensitive (ssl ^–) to G1 arrest by the mating hormone a-factor were isolated by screening mutagenized Saccharomyces cerevisiae MAT cells on solid medium for increased growth inhibition with a-factor. These mutants carried lesions in two complementation groups, ssl1 and ssl2. Mutations at the ssl1 locus were mating type specific: MAT ssl1 ^– cells were supersensitive to -factor but MAT ssl1 ^– were not supersensitive to -factor. In contrast, mutations at the ssl2. locus conferred supersensitivity to the mating hormone of the opposite mating type on both MAT, and MATa cells. The -cell specific capacity to inactivate externally added a-factor was shown to be lacking in MAT ssl1 ^– mutants whereas MAT ssl2. cells were able to inactivate a-factor. Complementation analysis showed that ssl2 and sst2, a mutation originally isolated as conferring supersensitivity to -factor to MATa cells, are lesions in the same gene. The ssl1 gene was mapped 30.5 centi-Morgans distal to ilv5 on chromosome XII.     

Effects of seed manganese concentration on lupin emergence

Seed of narrow-leafed lupin (Lupinus angustifolius L.) produced in Western Australia often has low manganese (Mn) concentration because of low Mn availability in the soil during grain filling. A major problem of lupin production is poor seedling establishment. We tested the hypothesis that low Mn concentration in lupin seeds decreases emergence.The experiment was a factorial design comparing emergence of lupins (cv. Gungurru) grown under glasshouse conditions from seed with 2 different internal Mn concentrations (7 or 35 mg Mn kg^–1 DW) and with 2 external Mn fertiliser treatments (0 or 10 mg MnSO₄.H₂O kg^–1 soil). There were no visible differences between the seeds. Emergence was monitored and plants were harvested 17 days after sowing.Emergence was approximately 60% in all pots sown with low Mn compared to 100% in pots sown with high Mn seed. Application of Mn did not increase the final emergence of low Mn seed. Seed viability was assessed by staining with tetrazolium chloride, a common test used in seed testing laboratories. All high Mn seed were viable while 34% of low Mn seed were completely or partly unstained and therefore were non-viable. We have shown that low Mn supply during seed filling may lead to production of non-viable seed that cannot be visually distinguished from viable seed..     

Environmental variables for modeling wheat yields in the southwest pampa region of Argentina

Two types of time scales – 10-day intervals (D) and phenological phases (P) – were applied to environmental variables for the development of statistical regression models relating to the southwest pampa region of Argentina, with the aim of detecting the effects of weather on wheat yields for the period 1977–1999. The parameters were grouped as meteorological and processed variables and indices. The processed variables used were total soil water availability (SWA) and the ratio of actual evapotranspiration to potential evapotranspiration (), obtained from a water-balance model in which the moisture anomaly index (Z) and the Palmer drought severity index (PDSI) were calculated according to Palmers model. For these parameters it was possible to establish the times of year and the phenological phases with the best correlation to grain yield. The regression equation for meteorological variables on a 10-day scale provides one of the best fits. Using mixed parameters, the two models, D and P, give rise to a standard error of estimate of approximately 200 kg ha^–1. Truncated models perform better on a P scale than on a D scale. The use of phenological stages improved yield assessment, particularly for those years with extreme meteorological conditions. The optimum models were tested and root-mean-square errors (RMSE) of 440 kg ha^–1 and 470 kg ha^–1 were obtained for P and D scales respectively.     

α-Globin gene deletions associated with αAand αG Philadelphiagenes in an Algerian family that includes two Hb G homozygotes

Summary An Algerian family with a high degree of consanguinity and including two homozygotes for Hb-G Philadelphia is presented. Whether homozygotes or heterozygotes, all subjects displayed microcytosis (with various degrees of poikilocytosis) and a moderately depressed -globin chain synthesis. Hb H and Heinz bodies were absent. DNA mapping revealed the presence of 3.7 kb deletion resulting from the rightward type of recombination event between ₂ and ₁ genes on both the ^A/and the ^G chromosomes. Such data indicate that the –^A/ and –^G/ haplotypes are involved and suggest that the –^G/ haplotype, which is very rare in Algeria, has an African Black origin. In subjects with genotype (–^A/–^G/) or (–^G/–^G/), the output of the remaining genes is sufficiently high to avoid the appearance of Hb H. This situation contrasts with that reported in an Algerian patient, who had a (–^A/–^A/) genotype but who was producing Hb H (Whitelaw et al. 1980). The data collected from this family suggest that the –^A/ haplotypes are heterogeneous in Algerians.     

Microbial populations in trifluralin-treated soil

Summary This study examined the effects of trifluralin (,,-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine), a soil incorporated herbicide, on soil microflora both in the general soil environment and in the rhizosphere of trifluralin damaged wheat roots. Two Dark Brown Chernozemic soils were treated with various trifluralin rates in the growth chamber and wheat [Triticum aestivum L. Neepawa] was seeded. Trifluralin generally had no effect on fungi, bacteria, or actinomycete populations in either the general soil or in the rhizosphere. CO₂ evolution was unchanged when trifluralin was added to the soil. In wheat plots, at two field locations, there were no significant effects of trifluralin (1.0 kg ha^–1) on soil fungi, bacteria, actinomycete, denitrifying bacteria, and nitrifying Nitrobacter propulations. A pure culture study with 42 soil microorganisms showed that many isolates were inhibited at 400 to 100,000 g g^–1 but not at concentrations <16 g g^–1. Similar data were obtained from tests on four different soils. These studies indicate that trifluralin is unlikely to cause changes in the numbers of soil microorganisms when used at recommended levels.     

Absence of red-light enhancement of phototropism in pea seedlings at limiting irradiances of blue light

The effect of different light qualities (blue, green, white, red and far-red) on ethylene production in leaf discs and flower petal discs of Begonia × hiemalis cv. Schwabenland Red was studied. All the light qualities, except far-red, reduced the ACC-conversion to ethylene in leaf discs by about 70% at a photosynthetic photon flux density (PPFD) of 20 mol m^–2s^–1.Blue and green light were less inhibitory than white and red light at lower PPFD. In all treatments far-red light at 0.5 mol m^–2s^–1 of photon flux density (PFD) stimulated the ACC-conversion to ethylene in leaf discs by about 60–90% compared to the dark-incubated control. White and red light strongly inhibited the -naphthalene-acetic acid (NAA) stimulated ethylene synthesis in leaf discs. The results may suggest that the ethylene production is controlled by phytochrome in the leaves but not in the petals. Lack of coaction of any light quality with silver ions on ethylene production in leaf and petal discs was also observed.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - EFE ethylene forming enzyme - NAA -naphthalene-acetic acid - PFD photon flux density - PPFD photosynthetic photon flux density - RH relative air humidity - SAM S-adenosylmethionine - STS silver thiosulphate     

Isolation and characterization of α-amylase derived from starchgrownClostridium acetobutylicum ATCC 824

Summary An extracellular -amylase was purified to homogeneity from the culture supernatant ofClostridium acetobutylicum ATCC 824 grown in synthetic medium containing starch by using a combination of ammonium sulfate fractionation, anion exchange chromatography and HPLC-gel filtration. The molecular weight of the 160-fold purified -amylase was determined by SDS-PAGE to be 61 kDa. HPLC analysis of end-products of enzyme activity on various substrates indicated that the enzyme acted specifically in an endo-fashion on the -1,4-glucosidic linkages. Enzyme activity was optimal over a pH range of 4.5–5.0 and temperature of 55°C, but was rapidly inactivated at higher temperatures. Addition of calcium chloride (2–5 mM) increased -amylase activity by ca. 20%, while the addition of 19 g ml^–1 of acarbose (a differential inhibitor of amylases) resulted in 50% inhibition. TheV _max andK _m of -amylase were 2.17 mg min^–1 and 3.28 mg ml^–1 on amylose, and 1.67 mg min^–1 and 1.73 mg ml^–1 on soluble starch, respectively.     

Efficient and precise measurement of H(alpha)-C(alpha), C(alpha)-C', C(alpha)-C(beta) and H(N)-N residual dipolar couplings from 2D H(N)-N correlation spectra

A suite of experiments are presented for the measurement of H–C, C–C, C–C and H^N–N couplings from uniformly ¹⁵N, ¹³C labeled proteins. Couplings are obtained from a series of intensity modulated two-dimensional H^N–N spectra equivalent to the common ¹H–¹⁵N–HSQC spectra, alleviating many overlap and assignment issues associated with other techniques. To illustrate the efficiency of this method, H–C, C–C, and H^N–N isotropic scalar couplings were determined for ubiquitin from data collected in less than 4.5 h, C–C data collection required 10 h. The resulting couplings were measured with an average error of ±0.06, ±0.05, ±0.04 and ±0.10 Hz, respectively. This study also shows H–C and C–C couplings, valuable because they provide orientation of bond vectors outside the peptide plane, can be measured in a uniform and precise way. Superior accuracy and precision to existing 3D measurements for C–C couplings and increased precision compared to IPAP measurements for H^N–N couplings are demonstrated. Minor modifications allow for acquisition of modulated H^N–C 2D spectra, which can yield additional well resolved peaks and significantly increase the number of measured RDCs for proteins with crowded ¹H–¹⁵N resonances.     

Enhancement of plasmid stability and protein productivity using multi-pulse, fed-batch culture of recombinant Saccharomyces cerevisiae

The plasmid stability of a recombinant Saccharomyces cerevisiae strain, which expresses cloned -amylase, was increased when glucose or yeast extract was fed with multi-pulse mode in fed-batch culture. Using a novel strategy combining constant rate fed-batch culture and multi-pulse feeding of yeast extract, the plasmid stability was maintained over 90%, meanwhile, 36 g cells l^–1 and 208 units of cloned -amylase activity ml^–1 were obtained.     

In vitro plantlet formation from juice vesicle callus of satsuma (Citrus unshiu Marc.)

Callus was induced from juice vesicles of satsuma mandarin on Murashige & Skoog medium supplemented with -naphthaleneacetic acid (NAA), kinetin (K) and gibberellin (GA). Adventitious embryoids arose from the callus tissue on the medium containing 1 mgl^–1 NAA alone. The embryoids grew into embryos which resulted in a plantlet on medium containing 1 mgl^–1 GA.Abbreviations GA gibberellin - K kinetin - NAA -naphthaleneacetic acid     

Functional expression of keratinase (kerA) gene from Bacillus licheniformis in Pichia pastoris

A 1.2 kb DNA fragment coding for the pro-peptide and mature keratinase from Bacillus licheniformis PWD-1 (kerA) was cloned into vectors pPICZA and pGAPZA for extracellular expression in the methylotrophic yeast, Pichia pastoris. Recombinant keratinase was secreted by the pPICZA-kerA transformants 24 h after methanol induction of shake-flask cultures, and reached a final yield of 124 mg l^–1 (285 U ml^–1) 144 h after the induction. The recombinant keratinase was glycosylated ( 39 kDa), and was optimal between pH 8.5–9.5 and between 55°C –60°C using azokeratin as substrate. The enzyme degraded bovine serum albumin, collagen, and soy protein concentrate. In conclusion, P. pastoris can be used as an efficient host to express keratinase for nutritional and environmental applications.     

Tolerance to Diazepam-Induced Motor Impairment: A Study with GABAA Receptor α6 Subunit Knockout Mice

Development of tolerance to motor-impairing effects of repeated administration of moderate diazepam doses (5.0–7.5 mg/kg; three times daily PO 3 weeks) was compared between mice deficient in the cerebellar granule cell–restricted GABA_A receptor 6 subunit and their wild-type controls. The 6–/– mice were more impaired by the initial challenge doses of diazepam (5 or 10 mg/kg) than their controls, but acquired partial tolerance by the second tests with the same doses 4–7 days later. Chronic treatment produced complete tolerance in both mouse lines. Ligand autoradiography revealed a significant reduction in baseline benzodiazepine and chloride channel site-bindings in various regions of the 6–/– brains, but the chronic diazepam treatment did not consistently alter baseline or benzodiazepine site agonist and inverse agonist-modulated binding in the 6–/– and wildtype mice. The results indicate that tolerance to motor-impairing actions of diazepam is independent of the diazepam-insensitive 6 subunit-containing receptors, which rules out the possibility that tolerance emerges as an increase in structurally benzodiazepine-insensitive receptor population.     

Production of thermostable α-galactosidase from thermophilic fungusHumicola sp

The thermophilic fungus,Humicola sp isolated from soil, secreted extracellular -galactosidase in a medium cotaining wheat bran extract and yeast extract. Maximum enzyme production was found in a medium containing 5% wheat bran extract as a carbon source and 0.5% beef extract as a carbon and nitrogen source. Enzyme secretion was strongly inhibited by the presence of Cu²⁺, Ni²⁺ and Hg²⁺ (1mM) in the fermentation medium. Production of enzyme under stationary conditions resulted in 10-fold higher activity than under shaking conditions. The temperature range for production of the enzyme was 37° C to 55°C, with maximum activity (5.54 U ml^–1) at 45°C. Optimum pH and temperature for enzyme activity were 5.0 and 60° C respectively. One hundred per cent of the original activity was retained after heating the enzyme at 60°C for 1 h. At 5mM Hg²⁺ strongly inhibited enzyme activity. TheK _m andV _max forp-nitrophenyl--d-galactopyranoside were 60M and 33.6 mol min^–1 mg^–1, respectively, while for raffinose those values were 10.52 mM and 1.8 mol min^–1 mg^–1, respectively.     

Functional analysis of linker insertions and point mutations in the α-Amy2/54 GA-regulated promoter

Functional analysis of a gibberellin-regulated wheat -amylase promoter, -Amy2/54, has indicated that three regions were essential for expression. By studying the ability of mutant promoters, containing a randomly inserted 22 bp excision linker, to direct expression in oat aleurone protoplasts we have refined the positions and extents of these three cis elements and also demonstrated the presence of two additional elements. By converting the linker insertions to either single base point mutations or deletions using the class IIS restriction endonuclease Bsm I we have shown that nucleotides –119 and –109 within the GARE ^–121GTAACAGAGTCTGG^–108 and nucleotide –152 within the proposed element ^–156GATTGACTTGACC^–144 are essential for high level expression from this promoter.