High resolution <Superscript>13</Superscript>C-detected solid-state NMR spectroscopy of a deuterated protein

High resolution ¹³C-detected solid-state NMR spectra of the deuterated beta-1 immunoglobulin binding domain of the protein G (GB1) have been collected to show that all ¹⁵N, ¹³C′, ¹³Cα and ¹³Cβ sites are resolved in ¹³C–¹³C and ¹⁵N–¹³C spectra, with significant improvement in T ₂ relaxation times and resolution at high magnetic field (750 MHz). The comparison of echo T ₂ values between deuterated and protonated GB1 at various spinning rates and under different decoupling schemes indicates that ¹³Cα T ₂′ times increase by almost a factor of two upon deuteration at all spinning rates and under moderate decoupling strength, and thus the deuteration enables application of scalar-based correlation experiments that are challenging from the standpoint of transverse relaxation, with moderate proton decoupling. Additionally, deuteration in large proteins is a useful strategy to selectively detect polar residues that are often important for protein function and protein–protein interactions.     

Development of laboratory-scale photobioreactor for water purification by use of a biofilter composed of the aerial microalga Trentepohlia aurea (Chlorophyta)

Biofilms formed by the green alga Trentepohlia aurea could be a useful tool in the removal of nitrate and phosphate from water. When a prepared biofilter was dampened with medium and incubated under low light intensity (10 μmol photons m⁻² s⁻¹) between 5 and 50 μmol photons m⁻² s⁻¹, the efficiency of removal of inorganic compounds from water was higher without the decomposition of chlorophylls in the cells. Algal cells immobilized on a glass fiber filter could be kept for 12 weeks under dark conditions at 4°C in the refrigerator. We tried to construct a laboratory-scale photobioreactor for the removal of inorganic nitrogen and phosphate from water by the biofilm. In this study, the synthetic wastewater was prepared by diluting 18-fold Bold’s basal medium with deionized water. The photobioreactor could efficiently remove nitrate and phosphate from the synthetic wastewater under continuous illumination. The removal ability of nitrate and phosphate per sheet of the biofilter in the photobioreactor exhibited about an 8- and 16-fold increase, respectively, in 3 days, compared with the bath experimental results. This study showed that the cycling of wastewater in the reactor by the pump led to a significant improvement in the efficiency of the inorganic ion uptake from water.     

Net Ecosystem Exchange of Carbon dioxide in a Temperate Poor Fen: a Comparison of Automated and Manual Chamber Techniques

We used five analytical approaches to compare net ecosystem exchange (NEE) of carbon dioxide (CO₂) from automated and manual static chambers in a peatland, and found the methods comparable. Once per week we sampled manually from 10 collars with a closed chamber system using a LiCor 6200 portable photosynthesis system, and simulated four photosynthetically active radiation (PAR) levels using shrouds. Ten automated chambers sampled CO₂ flux every 3 h with a LiCor 6252 infrared gas analyzer. Results of the five comparisons showed (1) NEE measurements made from May to August, 2001 by the manual and automated chambers had similar ranges: −10.8 to 12.7 μmol CO₂ m⁻² s⁻¹ and −17.2 to 13.1 μmol CO₂ m⁻² s⁻¹, respectively. (2) When sorted into four PAR regimes and adjusted for temperature (respiration was measured under different temperature regimes), mean NEE did not differ significantly between the chambers (p < 0.05). (3) Chambers were not significantly different in regression of ln( − respiration) on temperature. (4) But differences were found in the PAR vs. NEE relationship with manual chambers providing higher maximum gross photosynthesis estimates (GP_max), and slower uptake of CO₂ at low PAR (α) even after temperature adjustment. (5) Due to the high variability in chamber characteristics, we developed an equation that includes foliar biomass, water table, temperature, and PAR, to more directly compare automated and manual NEE. Comparing fitted parameters did not identify new differences between the chambers. These complementary chamber techniques offer a unique opportunity to assess the variability and uncertainty in CO₂ flux measurements.     

Survivorship of a monocarpic bamboo grass,Sasa kurilensis,during the early regeneration process after mass flowering

The survivorship of a monocarpic bamboo grass,Sasa kurilensis, during the early regeneration process was documented by a 10 year observation of the seedling population after mass flowering in the Hakkoda Mountains, northern Japan. Three phases were recognized: the establishment, density-stable and thinning phases. The mortality of the densely germinated seedlings (932.9m⁻² in aBetula ermanii forest and 1222.3 m⁻² in aSasa grassland) was high, up to 0.5 year⁻¹, in the establishment phase (0–1 year after germination) and low in the density-stable phase (1–3 years after germination). After reaching full density state, the seedling population showed a nearly constant mortality of 0.18 year⁻¹ due to self-thinning (the thinning phase). The high C/F ratio presumably caused suppressed seedlings to die. Recovery of theS. kurilensis population was estimated to requireca 20 years in the study plots, judging from the height growth and the decrease in culm density of the seedling population. The illuminance on the ground was higher in the flowered population than in the unflowered one for 5 years after mass death. The duration of high ground illuminance is an important factor affecting the dynamics of forests withSasa undergrowth, because tree seedlings need to establish under high ground illuminance for the successful regeneration of the forests.     

The Role of MIP in Lens Fiber Cell Membrane Transport

MIP has been hypothesized to be a gap junction protein, a membrane ion channel, a membrane water channel and a facilitator of glycerol transport and metabolism. These possible roles have been indirectly suggested by the localization of MIP in lens gap junctional plaques and the properties of MIP when reconstituted into artificial membranes or exogenously expressed in oocytes. We have examined lens fiber cells to see if these functions are present and whether they are affected by a mutation of MIP found in Cat ^Fr mouse lens. Of these five hypothesized functions, only one, the role of water channel, appears to be true of fiber cells in situ. Based on the rate of volume change of vesicles placed in a hypertonic solution, fiber cell membrane lipids have a low water permeability (p _H2O ) on the order of 1 μm/sec whereas normal fiber cell membrane p _H2O was 17 μm/sec frog, 32 μm/sec rabbit and 43 μm/sec mouse. Cat ^Fr mouse lens fiber cell p _H2O was reduced by 13 μm/sec for heterozygous and 30 μm/sec for homozygous mutants when compared to wild type. Lastly, when expressed in oocytes, the p _H2O conferred by MIP is not sensitive to Hg²⁺ whereas that of CHIP28 (AQP1) is blocked by Hg²⁺. The fiber cell membrane p _H2O was also not sensitive to Hg²⁺ whereas lens epithelial cell p _H2O (136 μm/sec in rabbit) was blocked by Hg²⁺. With regard to the other hypothesized roles, fiber cell membrane or lipid vesicles had a glycerol permeability on the order of 1 nm/sec, an order of magnitude less than that conferred by MIP when expressed in oocytes. Impedance studies were employed to determine gap junctional coupling and fiber cell membrane conductance in wild-type and heterozygous Cat ^Fr mouse lenses. There was no detectable difference in either coupling or conductance between the wild-type and the mutant lenses. Received: 17 February 1999/Revised: 16 April 1999     

Production of raw-starch-hydrolysing α-amylase from the newly isolated Geobacillus thermodenitrificans HRO10

An extracellular raw-starch-digesting α-amylase was isolated from Geobacillus thermodenitrificans HRO10. The culture conditions for the production of α-amylase by G. thermodenitrificans HRO10 was optimized in 1.2–l bioreactor using full 2⁴ and 3² factorial designs. From the optimal reaction conditions, a model (Y = − 594.206 − 0.178T² − 8.448pH² + 6.020TpH − 0.005T²pH²) was predicted, which was then used for α-amylase production. In the bioreactor studies, the enzyme yield under optimized conditions (pH 7.1, 49°C) was 30.20 U/ml, a 51% improvement over the results (19.97 U/ml) obtained when the traditional one-factor-at-a-time method was employed. This α-amylase does not require extraneous calcium ions for activity, which may be a commercially important observation.     

Effects of fluvalinate on two-spotted spider mite dispersal, fecundity and feeding

Residual deposits of fluvalinate caused two-spotted spider mite,Tetranychus urticae Koch, dispersal by running off (run-off) and spinning down (spin-down) from treated leaves. Run-off occured at concentrations from 0.0005 g a.i. 1⁻¹ and reached a peak at 0.01 g a.i. 1⁻¹. Spin-down was more predominant at concentrations above 0.01 g a.i. 1⁻¹. Spin-down and run-off proportions varied with fluvalinate formulation. Oviposition was temporarily suppressed on fluvalinate-treated leaves, presumably because of an irritant effect which reduced feeding. An emulsifiable concentrate formulation caused the largest reductions in oviposition. Oviposition returned to the level exhibited by untreated mites two days after removal from treated surfaces. Oviposition and feeding were positively correlated and both declined reciprocally with increasing fluvalinate concentration.     

The development of antenna complexes of barley (Hordeum vulgare cv. Akcent) under different light conditions as judged from the analysis of 77 K chlorophyll a fluorescence spectra

Using 77 K chlorophyll a (Chl a) fluorescence spectra in vivo, the development was studied of Photosystems II (PS II) and I (PS I) during greening of barley under intermittent light followed by continuous light at low (LI, 50 μmol m⁻² s⁻¹) and high (HI, 1000 μmol m⁻² s⁻¹) irradiances. The greening at HI intermittent light was accompanied with significantly reduced fluorescence intensity from Chl b excitation for both PS II (F685) and PS I (F743), in comparison with LI plants, indicating that assembly of light-harvesting complexes (LHC) of both photosystems was affected to a similar degree. During greening at continuous HI, a slower increase of emission from Chl b excitation in PS II as compared with PS I was observed, indicating a preferred reduction in the accumulation of LHC II. The following characteristics of 77 K Chl a fluorescence spectra documented the photoprotective function of an elevated content of carotenoids in HI leaves: (1) a pronounced suppression of Soret region of excitation spectra (410–450 nm) in comparison with the red region (670–690 nm) during the early stage of greening indicated a strongly reduced excitation energy transfer from carotenoids to the Chl a fluorescing forms within PS I and PS II; (2) changes in the shape of the excitation band of Chl b and carotenoids (460–490 nm) during greening under continuous light confirmed that the energy transfer from carotenoids to Chl a within PS II remained lower as compared with the LI plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Anaerobic biodegradation of triphenylmethane dyes in a hybrid UASFB reactor for wastewater remediation

Anaerobic digestions have been proved more successful than aerobic systems for the degradation and destruction of dye-containing wastewaters. The performance of a hybrid up flow anaerobic sludge-filter bed (UASFB) reactor was tested with a synthetic wastewater containing Crystal violet (CV) as a carbon source and sodium acetate as a co-substrate. Continuous feeding of the reactor started with an initial OLR of 0.9 g COD/l-d and then it was increased step wise to 4 g COD l⁻¹ d⁻¹, while maintaining constant HRT (24 h). The optimum pH value and temperature for decolorization of crystal violet by this mixed culture species under anaerobic conditions were found to be 8–9 and 30–35°C respectively. N,N-dimethylaminophenol and N,N-bis (dimethylamino) benzophenone (Michler’s Ketone) were detected as the degradative metabolites of Crystal Violet. Subsequently, N,N-dimethylaminophenol was further degraded to aniline in the reactor whereas Michler’s ketone was not degraded under anaerobic conditions. The UASFB bioreactor was able to remove the CV completely up to a loading rate of 100 mg CV l⁻¹d⁻¹.     

Complete stereospecific repair of a synthetic dinucleotide spore photoproduct by spore photoproduct lyase

Spore photoproduct lyase (SP lyase), a member of the radical S-adenosylmethionine superfamily of enzymes, catalyzes the repair of 5-thyminyl-5,6-dihydrothymine [spore photoproduct (SP)], a type of UV-induced DNA damage unique to bacterial spores. The anaerobic purification and characterization of Clostridium acetobutylicum SP lyase heterologously expressed in Escherichia coli, and its catalytic activity in repairing stereochemically defined synthetic dinucleotide SPs was investigated. The purified enzyme contains between 2.3 and 3.1 iron atoms per protein. Electron paramagnetic resonance (EPR) spectroscopy reveals an isotropic signal centered at g = 1.99, characteristic of a [3Fe–4S]⁺ cluster accounting for 3–4% of the iron in the sample. Upon reduction, a nearly axial signal (g = 2.03, 1.93 and 1.92) characteristic of a [4Fe–4S]⁺ cluster is observed that accounts for 34–45% of total iron. Addition of S-adenosylmethionine to the reduced enzyme produces a rhombic signal (g = 2.02, 1.93, 1.82) unique to the S-adenosyl-l-methionine complex while decreasing the overall EPR intensity. This reduced enzyme is shown to rapidly and completely repair the 5R diastereomer of a synthetic dinucleotide SP with a specific activity of 7.1 ± 0.6 nmol min⁻¹ mg⁻¹, whereas no repair was observed for the 5S diastereomer.     

Acidification of Soil in a Dry Land Winter Wheat-sorghum/corn-fallow Rotation in the Semiarid U.S. Great Plains

Soil pH is decreasing in many soils in the semiarid Great Plains of the United States under dry land no-till (NT) cropping systems. This study was conducted to determine the rate of acidification and the causes of the acidification of a soil cropped to a winter wheat (Triticum aestivum L.)-grain sorghum [Sorghum bicolor (L.) Moench]/corn (Zea mays L.)-fallow rotation (W-S/C-F) under NT. The study was conducted from 1989 to 2003 on soil with a long-term history of either continuous NT management [NT(LT)] (1962–2003) or conventional tillage (CT) (1962–1988) then converted to NT [NT(C)] (1989–2003). Nitrogen was applied as ammonium nitrate (AN) at a rate of 23 kg N ha⁻¹ in 1989 and as urea ammonium nitrate (UAN) at an average annual rate of 50 kg N ha⁻¹ from 1990 to 2003 for both NT treatments. Soil samples were collected at depth increments of 0–5, 5–10, 10–15, and 15–30 cm in the spring of 1989 and 2003. Acidification rates for the NT(LT) and NT(C) treatments were 1.13 and 1.48 kmol H⁺ ha⁻¹ yr⁻¹ in the 0–30 cm depth, respectively. The amount of CaCO₃ needed to neutralize the acidification is 57 and 74 kg ha⁻¹ yr⁻¹ for the NT(LT) and NT(C) treatments, respectively. A proton budget estimated by the Helyar and Porter [1989, Soil Acidity and Plant Growth, Academic Press] method indicated that NO₃⁻ leaching from the 30 cm depth was a primary cause of long-term acidification in this soil. Nitrate leaching accounted for 59 and 66% of the H⁺ from the acid causing factors for NT(LT) and NT(C) treatments, respectively. The addition of crop residues to the soil neutralized 62 and 47% of the acidity produced from the leaching of NO₃⁻, and 37 and 31% of the acid resulting from NO₃⁻ leaching and the other acid-causing constituents for the NT(LT) and NT(C) treatments, respectively. These results document that surface soils in dry land W-S/C-F rotations under NT are acidifying under current management practices. Improved management to increase nitrogen uptake efficiency from applied fertilizer would help reduce the rate of acidification. The addition of lime materials to prevent negative impacts on grain yields may be necessary in the future under current management practices. A contribution of the university of Nebraska Agricultural Research Division, Lincoln, NE 68583. Journal series No. 15120     

Fine-mapping of qRL6.1, a major QTL for root length of rice seedlings grown under a wide range of NH4 + concentrations in hydroponic conditions

Root system development is an important target for improving yield in cereal crops. Active root systems that can take up nutrients more efficiently are essential for enhancing grain yield. In this study, we attempted to identify quantitative trait loci (QTL) involved in root system development by measuring root length of rice seedlings grown in hydroponic culture. Reliable growth conditions for estimating the root length were first established to renew nutrient solutions daily and supply NH₄ ⁺ as a single nitrogen source. Thirty-eight chromosome segment substitution lines derived from a cross between ‘Koshihikari’, a japonica variety, and ‘Kasalath’, an indica variety, were used to detect QTL for seminal root length of seedlings grown in 5 or 500 μM NH₄ ⁺. Eight chromosomal regions were found to be involved in root elongation. Among them, the most effective QTL was detected on a ‘Kasalath’ segment of SL-218, which was localized to the long-arm of chromosome 6. The ‘Kasalath’ allele at this QTL, qRL6.1, greatly promoted root elongation under all NH₄ ⁺ concentrations tested. The genetic effect of this QTL was confirmed by analysis of the near-isogenic line (NIL) qRL6.1. The seminal root length of the NIL was 13.5–21.1% longer than that of ‘Koshihikari’ under different NH₄ ⁺ concentrations. Toward our goal of applying qRL6.1 in a molecular breeding program to enhance rice yield, a candidate genomic region of qRL6.1 was delimited within a 337 kb region in the ‘Nipponbare’ genome by means of progeny testing of F₂ plants/F₃ lines derived from a cross between SL-218 and ‘Koshihikari’.     

Isolation and characterization of a family VII esterase derived from alluvial soil metagenomic library

A novel esterase gene, estDL30, was isolated from an alluvial metagenomic library using function-driven screening. estDL30 consisted of 1,524 nucleotides and encoded a 507-amino acid protein. Sequence analysis revealed that EstDL30 is similar to many type B carboxylesterases, containing a G-E-S-A-G pentapeptide with a catalytic Ser residue. Phylogenetic analysis suggested that EstDL30 belongs to the family VII lipases, together with esterases from Bacillus subtilis (P37967), Streptomyces coelicolor A3(2) (CAA22794), and Arthrobacter oxydans (Q01470). Purified EstDL30 showed its highest catalytic efficiency toward p-nitrophenyl butyrate, with a k _cat of 2293 s⁻¹ and k _cat/K _m of 176.4 s⁻¹mM⁻¹; however, little activity was detected when the acyl chain length exceeded C₈. Biochemical characterization of EstDL30 revealed that it is an alkaline esterase that possesses maximal activity at pH 8 and 40° C. The effects of denaturants and divalent cations were also investigated. EstDL30 tolerated well the presence of methanol and Tween 20. Its activity was strongly inhibited by 1 mM Cu²⁺ and Zn²⁺, but stimulated by Fe²⁺. The unique properties of EstDL30, its high activity under alkaline conditions and stability in the presence of organic solvents, may render it applicable to organic synthesis.     

Citric acid production from sucrose using a recombinant strain of the yeast Yarrowia lipolytica

The yeast Yarrowia lipolytica is able to secrete high amounts of several organic acids under conditions of growth limitation and carbon source excess. Here we report the production of citric acid (CA) in a fed-batch cultivation process on sucrose using the recombinant Y. lipolytica strain H222-S4(p67ICL1) T5, harbouring the invertase encoding ScSUC2 gene of Saccharomyces cerevisiae under the inducible XPR2 promoter control and multiple ICL1 copies (10–15). The pH-dependent expression of invertase was low at pH 5.0 and was identified as limiting factor of the CA-production bioprocess. The invertase expression was sufficiently enhanced at pH 6.0–6.8 and resulted in production of 127–140 g l⁻¹ CA with a yield Y _CA of 0.75–0.82 g g⁻¹, whereas at pH 5.0, 87 g l ⁻¹ with a yield Y _CA of 0.51 gg⁻¹ were produced. The CA-productivity Q _CA increased from 0.40 g l ⁻¹ h⁻¹ at pH 5.0 up to 0.73 g l ⁻¹ h⁻¹ at pH 6.8. Accumulation of glucose and fructose at high invertase expression level at pH 6.8 indicated a limitation of CA production by sugar uptake. The strain H222-S4(p67ICL1) T5 also exhibited a gene–dose-dependent high isocitrate lyase expression resulting in strong reduction (<5%) of isocitric acid, a by-product during CA production.     

Construction of a bacterial artificial chromosome library containing large Eco RI and Hin dIII genomic fragments of lettuce

 Existing bacterial artificial chromosome (BAC) vectors were modified to have unique EcoRI cloning sites. This provided an additional site for generating representative libraries from genomic DNA digested with a variety of enzymes. A BAC library of lettuce was constructed following the partial digestion of genomic DNA with HindIII or EcoRI. Several experimental parameters were investigated and optimized. The BAC library of over 50,000 clones, representing one to two genome equivalents, was constructed from six ligations; average insert sizes for each ligation varied between 92.5 and 142 kb with a combined average insert size of 111 kb. The library was screened with markers linked to disease resistance genes; this identified 134 BAC clones from four regions containing resistance genes. Hybridization with low-copy genomic sequences linked to resistance genes detected fewer clones than expected from previous estimates of genome size. The lack of hybridization to chloroplast and mitochondrial sequences demonstrated that the library was predominantly composed of nuclear DNA. The unique EcoRI site in the BAC vector should allow the integration of BAC cloning with other technologies that utilize EcoRI digestion, such as AFLP^TM markers and RecA-assisted restriction endonuclease (RARE) cleavage, to clone specific large EcoRI fragments from genomic DNA. Received: 5 August 1996 / Accepted: 23 August 1996     

Genetic transformation of a Rhizomucor pusillus mutant defective in asparagine-linked glycosylation: production of a milk-clotting enzyme in a less-glycosylated form

Rhizomucor pusillus 1116R3 has a defect in alg2 encoding a mannosyltransferase in the asparagine (N)-linked oligosaccharide biosynthetic pathway and produces proteins in less-glycosylated forms. For development of a genetic transformation system for this zygomycete, an uracil auxotroph (mutant 1116U17) as the host strain was derived by ultraviolet (UV) mutagenesis as 5-fluoroorotic acid-resistant colonies and the orotidine-5′-monophosphate (OMP) decarboxylase (pyr4) gene as a selection marker was cloned from the wild-type strain R. pusillus F27 by the polymerase chain reaction with primers designed on the basis of the pyr4 sequences from other fungi. The amino acid sequence of R. pusillus Pyr4 deduced from the nucleotide sequence showed high homology with the OMP decarboxylases from various fungi. The pyr4 gene on pUC19 (plasmid pRPPyr4) was introduced into protoplasts of R. pusillus 1116U17 by polyethylene glycol-assisted transformation. Transformation under optimized conditions yielded 5 Ura⁺ transformants with 1 μg pRPPyr4 DNA and 1 × 10⁷ viable protoplasts. Southern blot analysis of the genomic DNA from the transformants showed that multiple copies of the pRPPyr4 sequence were integrated into the genome by homologous recombination at the pyr4 locus. For the purpose of production of a milk-clotting aspartic proteinase (MPP) in a less-glycosylated form, mpp from the wild-type strain was cloned in pRPPyr4 and introduced into protoplasts of R. pusillus 1116U17. Transformants obtained in this way contained multiple copies of mpp at the chromosomal mpp locus and produced MPP as a mixture of molecules having no sugar chains and Man_0∼1GlcNAc₂ at the two N-linked glycosylation sites in an amount about 12 times larger than the parent strain. The transformation system for R. pusillus 1116U17 would be useful for production of proteins with truncated N-linked oligosaccharide chains. Received: 1 February 1999 / Received revision: 26 February 1999 / Accepted: 20 March 1999     

Long-term production of soluble human Fas ligand through immobilization of <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis> in a fibrous bed bioreactor

The production of recombinant glycoproteins in Dictyostelium discoideum by conventional cell culture methods was limited by low cell density as well as low growth rate. In this work, cotton towel with a good adsorption capability for D. discoideum cells was used as the immobilization matrix in an external fibrous bed bioreactor (FBB) system. With batch cultures in the FBB, the concentration of immobilized cells in the cotton fiber carrier increased to 1.37 × 10⁸ cells per milliliter after 110-h cultivation, which was about tenfold higher than the maximal cell density in the conventional free-cell culture. Correspondingly, a high concentration of soluble human Fas ligand (hFasL; 173.7 μg l⁻¹) was achieved with a high productivity (23 μg l⁻¹ h⁻¹). The FBB system also maintained a high density of viable cells for hFasL production during repeated-batch cultures, achieving a productivity of 9∼10 μg l⁻¹ h⁻¹ in all three batches studied during 15 days. The repeated-batch culture using immobilized cells of D. discoideum in the FBB system thus provides a good method for long-term and high-level production of hFasL.     

Purification and characterization of a new L-methioninase from solid cultures of <Emphasis Type="Italic">Aspergillus flavipes</Emphasis>

L-Methioninase was purified to electrophoretic homogeneity from cultures of Aspergillus flavipes using anion-exchange and gel filtration chromatography by 12.1 fold compared to the crude enzyme preparation. The purified enzyme had a molecular mass of 47 kDa under denaturing conditions and an isoelectric point of 5.8 with no structural glycosyl residues. The enzyme had optimum activity at pH 7.8 and pH stability from 6.8–8.0 at 35°C. The enzyme appeared to be catalytically stable below 40°C. The enzyme activity was strongly inhibited by DL-propargylglycine, hydroxylamine, PMSF, 2-mercaptoethanol, Hg⁺, Cu²⁺, and Fe²⁺, with slight inhibition by Triton X-₁₀₀. A flavipes L-methioninase has a higher catalytic affinity towards L-methionine (Km, 6.5 mM and Kcat, 14.1 S⁻¹) followed by a relative demethiolating activity to L-homo-cysteine (Km, 12 mM and Kcat, 9.3 S⁻¹). The enzyme has two absorption maxima at 280 and 420 nm, typical of other PLP-enzymes. Apo-L-methioninase has the ability to reconstitute its structural catalytic state completely upon addition of 0.15 mM PLP. L-Methioninase has neither an appreciable effect on liver function, platelet aggregation, nor hemolysis of human blood. The purified L-methioninase from solid cultures of A. flavipes displayed unique biochemical and catalytic properties over the currently applied Pseudomonad enzyme.     

Purification and characterization of a membrane-bound hydrogenase from Sporomusa sphaeroides involved in energy-transducing electron transport

Hydrogenase was solubilized from the cytoplasmic membrane fraction of betaine-grown Sporomusa sphaeroides, and the enzyme was purified under oxic conditions. The oxygen-sensitive enzyme was partially reactivated under reducing conditions, resulting in a maximal activity of 19.8 μmol H₂ oxidized min^–1 (mg protein)^–1 with benzyl viologen as electron acceptor and an apparent K _m value for H₂ of 341 μM. The molecular mass of the native protein estimated by native PAGE and gel filtration was 122 and 130 kDa, respectively. SDS-PAGE revealed two polypeptides with molecular masses of 65 and 37 kDa, present in a 1:1 ratio. The native protein contained 15.6 ± 1.7 mol Fe, 11.4 ± 1.4 mol S^2–, and 0.6 mol Ni per mol enzyme. The hydrogenase coupled with viologen dyes, but not with other various artificial electron carriers, FAD, FMN, or NAD(P)⁺. The amino acid sequence of the N-termini of the subunits showed a high degree of similarity to eubacterial membrane-bound uptake hydrogenases. Washed membranes catalyzed a H₂-dependent cytochrome b reduction at a rate of 0.18 nmol min^–1 (mg protein)^–1. Received: 7 September 1995 / Accepted: 4 December 1995     

A unique genome-wide association analysis in extended Utah high-risk pedigrees identifies a novel melanoma risk variant on chromosome arm 10q

Only two genome-wide association (GWA) screens have been published for melanoma (Nat Genet 47:920–925, 2009; Nat Genet 40:838–840, 2008). Using a unique approach, we performed a genome-wide association study in 156 related melanoma cases from 34 high-risk Utah pedigrees. Genome-wide association analysis was performed on nearly 500,000 markers; we compared cases to 2,150 genotypically matched samples from Illumina’s iControls database. We performed genome-wide association with EMMAX software, which is designed to account for population structure, including relatedness between cases. Three SNPs exceeded a genome-wide significance threshold of p < 5 × 10⁻⁸ on chromosome arm 10q25.1 (rs17119434, rs17119461, and rs17119490), where the most extreme p value was 7.21 × 10⁻¹². This study represents a new and unique approach to predisposition gene identification; and it is the first genome-wide association study performed in related cases in high-risk pedigrees. Our approach illustrates an example of using high-risk pedigrees for the identification of new melanoma predisposition variants.