Long-term decomposed straw return positively affects the soil microbial community.

AIMS: In order to understand the response of soil microbial communities to the long-term of decomposed straw return, the modifications of soil microbial community structure and composition induced by more than 10 years of fresh and decomposed straw return was investigated and the key environmental factors were analysed. METHODS AND RESULTS: Phospholipid fatty acid analysis and high-through sequencing technique were applied to analyse the structure and composition of the soil microbial communities. Compared with fresh straw, returning decomposed straw increased the relative abundance of bacteria and fungi by 1·9 and 7·7% at a rate of ~3750 kg ha-1 , and increased by 23·1 and 5·7%, at a rate of ~7500 kg ha-1 respectively. The relative abundance of the bacteria related to soil nitrification increased, but the ones related to soil denitrification decreased with decomposed straw return, which led to higher total nitrogen contents in soils. Moreover, returning decomposed straw reduced pathogenic fungal populations (genus of Alternara), which had significantly positive correlation with soil electric conductivity. It indicated that the long-term of decomposed straw return might have lower risk of soil-borne disease mainly for the reasonable soil salinity. CONCLUSIONS: Long-term of decomposed straw return could provide suitable nutrient and salinity for healthier development of soil microbial community, both in abundance and structure, compared with fresh straw return. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of the study helps to better understand how the microbial community modifications induced by decomposed straw return benefit on soil health. The obtained key factors impacting soil microbial community variations is meaningful in soil health management under conditions of straw return.

Uptake and distribution of N, P and heavy metals in three dominant salt marsh macrophytes from Yangtze River estuary, China.

We examined the variation in aboveground biomass accumulation and tissue concentrations of nitrogen (N), phosphorus (P), copper (Cu), zinc (Zn) and lead (Pb) in Phragmites australis (common reed), Spartina alterniflora (salt cordgrass), and Scirpus mariqueter throughout the growing season (April-October 2005), in order to determine the differences in net element accumulation and distribution between the three salt marsh macrophytes in the Yangtze River estuary, China. The aboveground biomass was significantly greater in the plots of S. alterniflora than in the plots of P. australis and S. mariqueter throughout the growing season (P<0.05). In August, the peak aboveground biomass was 1246+/-89 gDW/m(2), 2759+/-250 gDW/m(2) and 548+/-54 gDW/m(2) for P. australis, S. alterniflora and S. mariqueter, respectively. The concentrations of nutrients and heavy metals in plant tissues showed similar seasonal patterns. There was a steady decline in element concentrations of the aboveground tissues from April to October. Relative element concentrations in aboveground tissues were at a peak during the spring sampling intervals with minimum levels during the fall. But the concentrations of total nitrogen and total phosphorus in the belowground tissues were relatively constant throughout growing season. Generally, trace metal concentrations in the aboveground tissues of S. mariqueter was the highest throughout the growing season, and the metal concentrations of S. alterniflora tissues (aboveground and belowground) were greater than those of P. australis. Furthermore, the aboveground pools of nutrients and metals were consistently greater for S. alterniflora than for P. australis and S. mariqueter, which suggested that the rapid replacement of native P. australis and S. mariqueter with invasive S. alterniflora would significantly improve the magnitude of nutrient cycling and bioavailability of trace metals in the salt marsh and maybe transport more toxic metals into the water column and the detrital food web in the estuary.

NADPH-cytochrome P450 oxidoreductase. Structural basis for hydride and electron transfer.

NADPH-cytochrome P450 oxidoreductase catalyzes transfer of electrons from NADPH, via two flavin cofactors, to various cytochrome P450s. The crystal structure of the rat reductase complexed with NADP(+) has revealed that nicotinamide access to FAD is blocked by an aromatic residue (Trp-677), which stacks against the re-face of the isoalloxazine ring of the flavin. To investigate the nature of interactions between the nicotinamide, FAD, and Trp-677 during the catalytic cycle, three mutant proteins were studied by crystallography. The first mutant, W677X, has the last two C-terminal residues, Trp-677 and Ser-678, removed; the second mutant, W677G, retains the C-terminal serine residue. The third mutant has the following three catalytic residues substituted: S457A, C630A, and D675N. In the W677X and W677G structures, the nicotinamide moiety of NADP(+) lies against the FAD isoalloxazine ring with a tilt of approximately 30 degrees between the planes of the two rings. These results, together with the S457A/C630A/D675N structure, allow us to propose a mechanism for hydride transfer regulated by changes in hydrogen bonding and pi-pi interactions between the isoalloxazine ring and either the nicotinamide ring or Trp-677 indole ring. Superimposition of the mutant and wild-type structures shows significant mobility between the two flavin domains of the enzyme. This, together with the high degree of disorder observed in the FMN domain of all three mutant structures, suggests that conformational changes occur during catalysis.

Differential contributions of NADPH-cytochrome P450 oxidoreductase FAD binding site residues to flavin binding and catalysis.

Transfer of reducing equivalents from NADPH to the cytochromes P450 is mediated by NADPH-cytochrome P450 oxidoreductase, which contains stoichiometric amounts of tightly bound FMN and FAD. Hydrogen bonding and van der Waals interactions between FAD and amino acid residues in the FAD binding site of the reductase serve to regulate both flavin binding and reactivity. The precise orientation of key residues (Arg(454), Tyr(456), Cys(472), Gly(488), Thr(491), and Trp(677)) has been defined by x-ray crystallography (Wang, M., Roberts, D. L., Paschke, R., Shea, T. M., Masters, B. S., Kim, J.-J. P. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 8411-8416). The current study examines the relative contributions of these residues to FAD binding and catalysis by site-directed mutagenesis and kinetic analysis. Mutation of either Tyr(456), which makes van der Waals contact with the FAD isoalloxazine ring and also hydrogen-bonds to the ribityl 4'-hydroxyl, or Arg(454), which bonds to the FAD pyrophosphate, decreases the affinity for FAD 8000- and 25,000-fold, respectively, with corresponding decreases in cytochrome c reductase activity. In contrast, substitution of Thr(491), which also interacts with the pyrophosphate grouping, had a relatively modest effect on both FAD binding (100-fold decrease) and catalytic activity (2-fold decrease), while the G488L mutant exhibited, respectively, 800- and 50-fold decreases in FAD binding and catalytic activity. Enzymic activity of each of these mutants could be restored by addition of FAD. Kinetic properties and the FMN content of these mutants were not affected by these substitutions, with the exception of a 3-fold increase in Y456S K(m)(cyt )(c) and a 70% decrease in R454E FMN content, suggesting that the FMN- and FAD-binding domains are largely, but not completely, independent. Even though Trp(677) is stacked against the re-face of FAD, suggesting an important role in FAD binding, deletion of both Trp(677) and the carboxyl-terminal Ser(678) decreased catalytic activity 50-fold without affecting FAD content.

Mechanistic studies on the reductive half-reaction of NADPH-cytochrome P450 oxidoreductase.

Site-directed mutagenesis has been employed to study the mechanism of hydride transfer from NADPH to NADPH-cytochrome P450 oxidoreductase. Specifically, Ser457, Asp675, and Cys630 have been selected because of their proximity to the isoalloxazine ring of FAD. Substitution of Asp675 with asparagine or valine decreased cytochrome c reductase activities 17- and 677-fold, respectively, while the C630A substitution decreased enzymatic activity 49-fold. Earlier studies had shown that the S457A mutation decreased cytochrome c reductase activity 90-fold and also lowered the redox potential of the FAD semiquinone (Shen, A., and Kasper, C. B. (1996) Biochemistry 35, 9451-9459). The S457A/D675N and S457A/D675N/C630A mutants produced roughly multiplicative decreases in cytochrome c reductase activity (774- and 22000-fold, respectively) with corresponding decreases in the rates of flavin reduction. For each mutation, increases were observed in the magnitudes of the primary deuterium isotope effects with NADPD, consistent with decreased rates of hydride transfer from NADPH to FAD and an increase in the relative rate limitation of hydride transfer. Asp675 substitutions lowered the redox potential of the FAD semiquinone. In addition, the C630A substitution shifted the pKa of an ionizable group previously identified as necessary for catalysis (Sem, D. S., and Kasper, C. B. (1993) Biochemistry 32, 11539-11547) from 6.9 to 7.8. These results are consistent with a model in which Ser457, Asp675, and Cys630 stabilize the transition state for hydride transfer. Ser457 and Asp675 interact to stabilize both the transition state and the FAD semiquinone, while Cys630 interacts with the nicotinamide ring and the fully reduced FAD, functioning as a proton donor/acceptor to FAD.

Role of Ser457 of NADPH-cytochrome P450 oxidoreductase in catalysis and control of FAD oxidation-reduction potential.

Site-directed mutagenesis of Ser457 of NADPH-cytochrome P450 oxidoreductase demonstrates that this residue plays a major role in both hydride transfer from NADPH to FAD and modulation of FAD redox potential. Substitution of Ser457 with alanine or cysteine decreases the rates of reduction of the substrates cytochrome c and potassium ferricyanide approximately 100-fold, while substitution with threonine produces a 20-fold decrease in activity. No changes are observed in k(m)NADPH, KiNADP+, or flavin content, indicating that these substitutions have no effect on cofactor binding but affect catalysis only. k(m)cyt c values are decreased in parallel with the observed decreases in the rates of the reductive half-reaction. Stopped-flow studies with the S457A mutant show a 100-fold decrease in the rate of flavin reduction. The primary deuterium isotope effect on Kcat for cytochrome c reduction increases from 2.7 for the wild-type enzyme to 9.0 for the S457A mutant, consistent with a change in the rate-determining step from NADP+ release in the wild-type enzyme to hydride transfer in the S457A mutant. The primary deuterium isotope effect on K1 for flavin reduction at high ionic strength (I = 535 mM) increases from 12.2 for the wild-type enzyme to > 20 for the S457A mutant, consistent again with an increase in the relative rate limitation of hydride transfer. Furthermore, anaerobic titration of S457A indicates that the redox potential of the FAD semiquinone has been decreased. Data presented in this study support the hypothesis that Ser457 is involved in hydrogen bonding interactions which stabilize both the transition state for hydride transfer and the reduced FAD.

Role of acidic residues in the interaction of NADPH-cytochrome P450 oxidoreductase with cytochrome P450 and cytochrome c.

Site-directed mutagenesis of the acidic clusters 207Asp-Asp-Asp209 and 213Glu-Glu-Asp215 of NADPH-cytochrome P450 oxidoreductase demonstrates that both cytochrome c and cytochrome P450 interact with this region; however, the sites and mechanisms of interaction of the two substrates are clearly distinct. Substitutions in the first acidic cluster did not affect cytochrome c or ferricyanide reductase activity, but substitution of asparagine for aspartate at position 208 reduced cytochrome P450-dependent benzphetamine N-demethylase activity by 63% with no effect on KP450m or KNADPHm. Substitutions in the second acidic cluster affected cytochrome c reduction but not benzphetamine N-demethylase or ferricyanide reductase activity. The E213Q enzyme exhibited a 59% reduction in cytochrome c reductase activity and a 47% reduction in KCyt cm under standard conditions (x0.27 M potassium phosphate, pH 7.7), as well as a decreased KCyt cm at every ionic strength and a shift of the salt dependence of cytochrome c reductase activity toward lower ionic strengths. The E214Q substitution did not affect cytochrome c reductase activity under standard conditions, but shifted the salt dependence of cytochrome c reductase activity toward higher ionic strengths. Measurements of the effect of ionic strength on steady-state kinetic properties indicated that increasing ionic strength destabilized the reductase-cytochrome c3+ ground state and reductase-cytochrome c transition state complexes for the wild-type, E213Q, and E214Q enzymes, suggesting the presence of electrostatic interactions involving Glu213 and Glu214 as well as additional residues outside this region. The ionic strength dependence of kcat/KCyt cm for the wild-type and E214Q enzymes is consistent with the presence of charge-pairing interactions in the transition state and removal of a weak ionic interaction in the reductase-cytochrome c transition-state complex by the E214Q substitution. The ionic strength dependence of the E213Q enzyme, however, is not consistent with a simple electrostatic model. Effects of ionic strength on kinetic properties of E213Q suggest that substitution of glutamine stabilizes the reductase-cytochrome c3+ ground-state complex, leading to a net increase in activation energy and decrease in kcat. Glu213 is also involved in a repulsive interaction with cytochrome c3+. Cytochrome c2+ Ki for the wild-type enzyme was 82.4 microM at 118 mM ionic strength and 10.8 microM at 749 mM ionic strength; similar values were observed for the E214Q enzyme. Cytochrome c Ki for the E213Q enzyme was 17.6 microM at 118 mM and 15.7 microM at 749 mM ionic strength, consistent with removal of an electrostatic repulsion between the reductase and cytochrome c2+.

NADPH-cytochrome P-450 oxidoreductase. The role of cysteine 566 in catalysis and cofactor binding.

Site-directed mutagenesis was employed to investigate the role of Cys566 in the catalytic mechanism of rat liver NADPH-cytochrome P-450 oxidoreductase. Rat NADPH-cytochrome P-450 oxidoreductase and mutants containing either alanine or serine at position 566 were expressed in Escherichia coli and purified to homogeneity. Substitution of alanine at position 566 had no effect on enzymatic activity with the acceptors cytochrome c and ferricyanide but did increase trans-hydrogenase activity with 3-acetylpyridine adenine dinucleotide phosphate by 79%. The Km for NADPH was increased 2.5-fold, and the NADP+ KI was increased 4.8-fold compared with that found for the wild-type enzyme. The conservative substitution, Ser566, produced a 50% decrease in cytochrome c reductase activity whereas activity with ferricyanide was decreased 57%, and 3-acetylpyridine adenine dinucleotide phosphate activity was unaffected. The NADPH Km was increased 4.6-fold, and the NADP+ KI increased 7.6-fold. The dependence of cytochrome c reductase activity on the KCl concentration was markedly altered by the Cys566 substitutions. Maximum activity for the wild-type enzyme was observed at approximately 0.18 M KCl whereas maximum activity for the mutant enzymes was observed between 0.04 and 0.09 M KCl. The pH dependence of cytochrome c reductase activity, cytochrome c Km, and flavin content were unaffected by these substitutions. These results demonstrate that Cys566 is not essential for activity of rat liver NADPH-cytochrome P-450 oxidoreductase although the cysteine side chain does affect the interaction of NADPH with the enzyme.

Structural analysis of the FMN binding domain of NADPH-cytochrome P-450 oxidoreductase by site-directed mutagenesis.

Comparison of the amino acid sequence of rat liver NADPH-cytochrome P-450 oxidoreductase with that of flavoproteins of known three-dimensional structure suggested that residues Tyr-140 and Tyr-178 are involved in binding of FMN to the protein. To test this hypothesis, NADPH-cytochrome P-450 oxidoreductase was expressed in Escherichia coli using the expression-secretion vector pIN-III-ompA3, and site-directed mutagenesis was employed to selectively alter these residues and demonstrate that they are major determinants of the FMN-binding site. Bacterial expression produced a membrane-bound 80-kDa protein containing 1 mol each of FMN and FAD per mol of enzyme, which reduced cytochrome c at a rate of 51.5 mumol/min/mg of protein and had absorption spectra and kinetic properties very similar to those of the rat liver enzyme. Replacement of Tyr-178 with aspartate abolished FMN binding and cytochrome c reductase activity. Incubation with FMN increased catalytic activity to a maximum of 8.6 mumol/min/mg of protein. Replacement of Tyr-140 with aspartate did not eliminate FMN binding, but reduced cytochrome c reductase activity about 5-fold, suggesting that FMN may be bound in a conformation which does not permit efficient electron transfer. Substitution of phenylalanine at either position 140 or 178 had no effect on FMN content or catalytic activity. The FAD level in the Asp-178 mutant was also decreased, suggesting that FAD binding is dependent upon FMN; FAD incorporation may occur co-translationally and require prior formation of an intact FMN domain.

The human immunoglobulin C mu-C delta locus: complete nucleotide sequence and structural analysis.

The entire nucleotide sequence (approximately 20 kbp) spanning the human immunoglobulin IgM (mu) and IgD (delta) heavy chain constant region genes has been determined from DNA of mu-delta producing chronic lymphocytic leukemic B cells. As in the murine IgM + IgD double-producing B cells, no rearrangement has occurred in the C mu-C delta region in the leukemic cells. The C mu locus is highly conserved between mouse and human with the exception of the nucleotide sequence between the C mu 4 and mu M1 exons, which has diverged dramatically. The intergenic sequence between human C mu and C delta is three times larger than the analogous region in the mouse and contains notable features absent from the mouse, including a 443 bp segment that is 96% identical to a 442 bp sequence that occurs just 3' to the heavy chain enhancer, a 366 bp sequence that is directly repeated with 76% homology, and 12 tandem copies of a 35 bp sequence. The human C delta gene contains two additional exons relative to mouse C delta, but shares with the mouse the unique distal location of both secreted and membrane coding segments. Several polymorphisms in the human population have been identified in the intergenic region and in C delta but not in C mu.

Cellular and antigenic properties of cultured normal and fetal brain and glioma cells.

The immunological and cellular properties of cultured normal and fetal brain cells as well as glioma cells were compared. They were grown successfully in tissue culture media. Results from the growth properties and karyotype analysis indicated that cultured cells from normal and fetal brain tissues were normal and could be passaged limited times. The fetal brain cells had a longer life span than normal brain cells in the culture and their morphology exhibited variations according to cell passages. Two glioma cell lines, designated as G-5-T and G-9-T were established. The G-5-T and G-9-T had different morphology. Both G-5-T and G-9-T formed colonies in the soft agar. However, only G-9-T cells grew as large tumors in nude mice. Neither cell line secreted CEA, AFP and did not contain GFAP and S-100 protein. As measured by the 51Cr cytotoxicity assay, G-9-T but not G-5-T cells possessed D/DR antigens.

Human immunoglobulin D: genomic sequence of the delta heavy chain.

The DNA coding for the human immunoglobulin D(IgD) heavy chain (delta, delta) has been sequenced including the membrane and secreted termini. Human delta, like that of the mouse, has a separate exon for the carboxyl terminus of the secreted form. This feature of human and mouse IgD distinguishes it from all other immunoglobulins regardless of species or class. The human gene is different from that of the mouse; it has three, rather than two, constant region domains; and its lengthy hinge is encoded by two exons rather than one. Except for the third constant region, the human and mouse genes are only distantly related.

Alterations in serum sialyltransferase activities in patients with brain tumors.

The activities of serum sialyltransferase were determined in patients with brain tumors. Blood samples from normal volunteers were used as controls. Serum specimens were obtained from patients with brain tumors both before and after operations. The preoperative serum sialyltransferase activities of the brain metastasis group showed significant increase, but the enzyme levels decreased after removal of the tumor. The serum sialyltransferase activities in the glioma group and the neurilemoma group increased significantly after operation, but no significant difference was found between the preoperative means of these two groups and that of the control. Surgical treatment produced significant differences between the preoperative and the postoperative serum sialyltransferase activities in the brain metastasis and the glioma and neurilemoma groups. Alterations of this enzyme in the blood of patients with brain tumors and its possible clinical applications are discussed.

Characterization of human growth hormone from acromegalic pituitary tumors.

Human growth hormone (HGH) was extracted from acromegalic pituitary tumors at pH 10.5 and precipitated with ammonium sulfate at 20-40% saturation. It was purified on a Sephadex G-100 column to yield monomeric HGH. The tumor-HGH was indistinguishable from the authentic one in polyacrylamide gel electrophoresis at pH 8.3 or in the presence of sodium dodecyl sulfate, high-performance liquid chromatography, radioimmunoassay, peptide map, amino acid composition and N-terminal partial amino acid sequence. The tumor-HGH is active in the tibia assay and bodyweight gain test in hypophysectomized rats with comparable potency to that of the authentic sample.

Hypoglossofacial anastomosis for facial palsy after resection of acoustic neuroma.

Twelve patients with facial palsy after resection of acoustic neuromas were treated by hypoglossofacial anastomosis. The nerve anastomosis were performed 1-2 months after resection of the tumor in 10 cases (group A). Two cases (group B) had the anastomosis performed more than 2 years after damage to the facial nerve. All the cases had been followed for more than 9 months. The results of reinnervation of the paralyzed facial muscles were quite satisfactory, i.e., over 80% of the cases obtained a good functional recovery. Although there was a 2-month delay in functional recovery of group B patients, the final results were practically the same in both groups. Therefore, the effect of the duration of the paralysis of the facial nerve seems to be less important in facial nerve surgery than in nerve surgery of the extremities. The procedures of anastomosis of descendens hypoglossi to the distal stump to the hypoglossal in 7 of the 10 patients using the hypoglossal as the donor nerve was of little help in prevention or restoration of the hemiatrophy of the tongue. In spite of long-term inconvenience in speaking and eating after section of the hypoglossal, all the patients were able to make enough adjustments about 2-3 months after nerve surgery.