Titratable groups and soluble phenolic compounds as indicators of the digestibility of chemically treated roughages

Methods are presented which evaluate the efficiency of delignification in alkali-treated grass straws. Saponified lignin is soluble in neutral detergent solution and in phosphate buffer, and can be assayed by ultraviolet spectrophotometry of the solutions. Extraction of free phenolic material by means of neutral detergent or phosphate buffer is followed by determination of a saponification number (titratable groups) on the residue. Measurements of phenolic matter in the sodium hydroxide extract of the fibre residue allow assay of the residual, bound lignin in treated straws. The bound lignin is negatively correlated with digestibility of alkali-treated straw. The results provide further evidence that the negative effect of lignin upon digestibility of forages depends on covalent linkage between lignin and the carbohydrate residues that it protects.     

Alkali‐based AFEX pretreatment for the conversion of sugarcane bagasse and cane leaf residues to ethanol

Sugarcane is one of the major agricultural crops cultivated in tropical climate regions of the world. Each tonne of raw cane production is associated with the generation of 130 kg dry weight of bagasse after juice extraction and 250 kg dry weight of cane leaf residue postharvest. The annual world production of sugarcane is ～1.6 billion tones, generating 279 MMT tones of biomass residues (bagasse and cane leaf matter) that would be available for cellulosic ethanol production. Here, we investigated the production of cellulosic ethanol from sugar cane bagasse and sugar cane leaf residue using an alkaline pretreatment: ammonia fiber expansion (AFEX). The AFEX pretreatment improved the accessibility of cellulose and hemicelluloses to enzymes during hydrolysis by breaking down the ester linkages and other lignin carbohydrate complex (LCC) bonds and the sugar produced by this process is found to be highly fermentable. The maximum glucan conversion of AFEX pretreated bagasse and cane leaf residue by cellulases was ～85%. Supplementation with hemicellulases during enzymatic hydrolysis improved the xylan conversion up to 95–98%. Xylanase supplementation also contributed to a marginal improvement in the glucan conversion. AFEX‐treated cane leaf residue was found to have a greater enzymatic digestibility compared to AFEX‐treated bagasse. Co‐fermentation of glucose and xylose, produced from high solid loading (6% glucan) hydrolysis of AFEX‐treated bagasse and cane leaf residue, using the recombinant Saccharomyces cerevisiae (424A LNH‐ST) produced 34–36 g/L of ethanol with 92% theoretical yield. These results demonstrate that AFEX pretreatment is a viable process for conversion of bagasse and cane leaf residue into cellulosic ethanol. Biotechnol. Bioeng. 2010;107: 441–450. © 2010 Wiley Periodicals, Inc.     

噻虫嗪灌根对四种叶菜上害虫的防治效果及残留检测

为明确噻虫嗪灌根对叶菜类蔬菜害虫的防治效果及残留情况,采用1.04 mg/株、1.39 mg/株和2.08 mg/株噻虫嗪对苗期快菜、青梗菜、菠菜以及叶用莴苣进行灌根处理,并调查对其主要害虫的防治效果及产品中的残留量。结果表明,在噻虫嗪有效成分为1.04 mg/株、1.39 mg/株和2.08 mg/株处理浓度下,能够将4种叶菜上的虫口数控制在2头/株以下,且产品中噻虫嗪残留量在0.0015-1.504 mg/kg,符合国际标准。因此,采用噻虫嗪有效成分1.04-2.08 mg/株的药剂浓度在苗期对叶菜类蔬菜进行灌根,可有效控制蚜虫等害虫的危害,并保证残留量符合安全标准。     

Evidence that both arginine 102 and arginine 747 are involved in substrate binding to neutral endopeptidase (EC 3.4.24.11)

Neutral endopeptidase (EC 3.4.24.11, NEP) is a Zn-metallopeptidase involved in the degradation of biologically active peptides, notably the enkephalins and atrial natriuretic peptide. Recently, the structure of the active site of this enzyme has been probed by site-directed mutagenesis, and 4 amino acid residues have been identified, namely 2 histidines (His583 and His587), which act as zinc-binding ligands, a glutamate (Glu584) involved in catalysis, and an arginine residue (Arg102), suggested to participate in substrate binding. Site-directed mutagenesis has now been used to investigate the role of 4 other arginine residues (Arg408, Arg409, Arg659, and Arg747) that have been proposed as possible active site residues and to further analyze the role of Arg102. In each case, the arginine was replaced with a methionine, and both enzymatic activity and the IC50 values of several NEP inhibitors were measured for the mutated enzymes and compared to wild-type enzyme. The results suggest that 2 arginines, Arg102 and Arg747, could both be important for substrate and inhibitor binding. Arg747 seems to be positioned to interact with the carbonyl amide group of the P'1 residue and can be modified when the enzyme is treated with the arginine-specific reagents phenylglyoxal and butanedione. Arg102 could be positioned to interact with the free carboxyl group of a P'2 residue in some substrates and inhibitors and can be modified by phenylglyoxal but not by butanedione. The results could explain the dual dipeptidylcarboxypeptidase and endopeptidase nature of NEP.     

Outgrowth of pseudopodial cables induced by all-trans retinoic acid in micromere-derived cells isolated from sea urchin embryos

Cultured cells derived from micromeres of sea urchin embryos underwent pseudopodial cable growth without spicule rod formation in the presence of all-trans retinoic acid (tRA) or insulin. Pseudopodial cable growth caused by tRA or insulin was inhibited by genistein, a protein tyrosine kinase inhibitor. Phosphorylation of protein tyrosine residue was augmented in the cells treated with tRA or insulin and was inhibited by genistein. Probably, protein tyrosine kinase takes an indispensable part in signal transduction systems for tRA and insulin in these cells. In tRA-treated cells, augmentation of the phosphorylation of protein tyrosine residue was accompanied by an increase in the activity of protein tyrosine kinase and was inhibited by actinomycin D, inhibiting cable growth. Activation of this enzyme in tRA-treated cells probably depends on RNA synthesis. In insulin-treated cells, augmentation of tyrosine residue phosphorylation occurred without any appreciable change in this enzyme's activity and was hardly affected by actinomycin D. Phosphorylation of protein tyrosine residue seems to be activated by the binding of insulin to an insulin receptor. Pseudopodial cable growth in these cells treated with tRA or insulin was inhibited by wortmannin. Phosphatidylinositol 3 kinase probably participates in tRA and insulin signal transduction systems.     

The bottom line for prediction of residue solvent accessibility

A simple method of predicting residue solvent accessibilities in proteins is described, with the intention that it should be used as a baseline by which more sophisticated approaches to prediction can be judged. Comparison with existing methods of predicting residue burial reveals that their performance is often little better than that of the baseline method. The problem of comparing different prediction methods is shown to be complicated by the proliferation of different schemes for classifying residue burial.     

On the resolution of polypeptides by isoelectric focusing in polyacrylamide gels in the presence of urea and nonidet-p40.

Three model substances were used to test the resolving power of isoelectric focusing in polyacrylamide gels in the presence of 9 m urea and 2% Nonidet-P40: (1) The α subunit of RNA polymerase from Escherichia coli modified by a single covalently attached adenosine 5′-diphosphate-ribose residue can be clearly resolved from the unmodified subunit. (2) The α subunit of RNA polymerase from a mutant of E. coli in which a single leucine residue is replaced by a histidine residue can be resolved from the wild-type subunit. (3) Muscle actin presumably modified by carboxymethylation with iodoacetate at only one cysteine residue can be separated from the unmodified polypeptide.     

Elucidation of the primary structures of the cockroach hyperglycaemic hormones I and II using enzymatic techniques and gas-phase sequencing

ABSTRACT. Two peptides, HGHI and HGHII, both inducing hyperglycaemia and activation of fat body glycogen phosphorylase can be isolated from the corpora cardiaca of the American cockroach, Periplaneta americana , using high-performance liquid chromatography. Both peptides are N-terminally blocked by a pyroglutamate residue and are thus not available for sequencing methods using the Edman degradation as this technique requires a free N-terminus. The blocked peptides were treated with pyroglutamate aminopeptidase to cleave the pyroglutamate residue, and the C-terminus of each peptide is also blocked and neither molecule can be cleaved by carboxypeptidase A. The following sequences for hyperglycaemic hormones HGHI and HGHII have been revealed using gas-phase sequencing.
HGHI pGlu-Val-Asn-Phe-Ser-Pro-Asn-Trp-NH₂
HGHII pGlu-Leu-Thr-Phe-Thr-Pro-Asn-Trp-NH₂
Both peptides show remarkable similarities to locust adipokinetic hormones I and II and prawn red-pigment concentrating hormone, and are identical to two myotropic peptides MI and MII (O'Shea et al. , 1984; Witten et al. , 1984) and two cardioactive and hyperglycaemic peptides CC-1 and CC-2 (Scarborough et al. , 1984), respectively, also isolated from the corpora cardiaca of P. americana.     

Monomeric creatine kinase aggregation and sodium dodecyl sulfate-cyclodextrin assisted refolding

The monomeric state of creatine kinase (CK) was stably captured at the equilibrium state by employing cysteine residue modifications in the presence of a denaturant, and at a partially folded state. The partially folded monomeric CK (PF-CK) was aggregated with kinetic order, which was mainly caused by the hydrophobic surface interactions between the CK subunits. The artificial chaperone, described as a SDS-cyclodextrin, was applied to prevent aggregation as well as to refold the PF-CK: SDS treatment onto the monomeric CK can significantly block aggregation and can be successfully refolded in the solutions containing cyclodextrins and DTT. Three types of cyclodextrins such as alpha-, beta-, and gamma-cyclodextrins were applied to strip SDS from the enzyme molecule, and each kinetic course was measured. The intrinsic fluorescence changes showed that reactivation occurred and this accompanied the conformational changes. The size exclusion chromatography detected the variously trapped monomeric CKs such as the thiol residue modified PF-CK, the SDS-binding PF-CK, the cyclodextrin treated PF-CK, and the DTT treated SDS-binding PF-CK. Our study demonstrated monomer CK aggregation for the first time; we also demonstrated the complex reassociation of CK during refolding with the aid of the SDS-cyclodextrin, and these pathways followed first-order kinetics.     

Cross-linking between 16S ribosomal RNA and protein S4 in Escherichia coli ribosomal 30S subunits effected by treatment with bisulfite/hydrazine and bromopyruvate

Cytosine in nucleic acids can be modified by treatment with a mixture of bisulfite and hydrazine. The reaction is specific for single-stranded regions of nucleic acids and the product is N4-aminocytosine. Bromopyruvate has been used for alkylation of protein SH groups and through its 2-oxo group it can form a hydrazone with N4-aminocytosine. Escherichia coli ribosomal 30S subunits were treated with 1 M sodium bisulfite + 2 M hydrazine in the presence of 10 mM MgCl2 at pH 7.0 and 37 degrees C for 30 min. By this treatment, 2.4 cytosine residues/molecule 16S rRNA were derivatized into N4-aminocytosines. 35S-labeled 30S subunits were modified in this way and then treated with 10 mM bromopyruvate at pH 8.0 and 37 degrees C for 5 min. Analysis in sodium dodecyl sulfate/sucrose density gradient centrifugation showed co-sedimentation of a part of the 35S radioactivity with the RNA. The co-sedimentation was dependent on both the bisulfite/hydrazine and the bromopyruvate treatments. The RNA-protein complex was prepared from unlabeled 30S subunits. The protein portion was labeled with 125I, the RNA portion was digested with nucleases, and then the hydrazone linkage between the protein and oligonucleotides was cleaved by treatment with 0.2 M HCl. The oligonucleotides formed were removed by dialysis and the protein was identified as S4 by two-dimensional electrophoresis and by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The results indicate that the cysteinyl residue of protein S4 at position 31 from the N-terminus is located close to a cytosine residue which is non-base-paired and easily accessible by the externally present bisulfite/hydrazine reagent.     

Structural characterization of the pectic polysaccharide rhamnogalacturonan II: evidence for the backbone location of the aceric acid-containing oligoglycosyl side chain

Monomeric rhamnogalacturonan II (mRG-II) was isolated from red wine and the reducing-end galacturonic acid of the backbone converted to L-galactonic acid by treatment with NaBH4. The resulting product (mRG-II'ol) was treated with a cell-free extract from Penicillium daleae, a fungus that has been shown to produce RG-II-fragmenting glycanases. The enzymatically generated products were fractionated by size-exclusion and anion-exchange chromatographies and the quantitatively major oligosaccharide fraction isolated. This fraction contained structurally related oligosaccharides that differed only in the presence or absence of a single Kdo residue. The Kdo residue was removed by acid hydrolysis and the resulting oligosaccharide then characterized by 1- and 2D 1H NMR spectroscopy, ESMS, and by glycosyl-residue and glycosyl-linkage composition analyses. The results of these analyses provide evidence for the presence of at least two structurally related oligosaccharides in the ratio approximately 6:1. The backbone of these oligosaccharides is composed of five (1-->4)-linked alpha-D-GalpA residues and a (1-->3)-linked L-galactonate. The (1-->4)-linked GalpA residue adjacent to the terminal non-reducing GalpA residue of the backbone is substituted at O-2 with an apiosyl-containing side chain. Beta3-L-Araf-(1-->5)-beta-D-DhapA is likely to be linked to O-3 of the GalpA residue at the non-reducing end of the backbone in the quantitatively major oligosaccharide and to O-3 of a (1-->4)-linked GalpA residue in the backbone of the minor oligosaccharide. Furthermore, the results of our studies have shown that the enzymically generated aceryl acid-containing oligosaccharide contains an alpha-linked aceryl acid residue and a beta-linked galactosyl residue. Thus, the anomeric linkages of these residues in RG-II should be revised.     

Kinetic analysis of the role of lipoic acid residues in the pyruvate dehydrogenase multienzyme complex of Escherichia coli

The catalytic roles of the two reductively acetylatable lipoic acid residues on each lipoate acetyltransferase chain of the pyruvate dehydrogenase complex of Escherichia coli were investigated. Both lipoyl groups are reductively acetylated from pyruvate at the same apparent rate and both can transfer their acetyl groups to CoASH, part-reactions of the overall complex reaction. The complex was treated with N-ethylmaleimide in the presence of pyruvate and the absence of CoASH, conditions that lead to the modification and inactivation of the S-acetyldihydrolipoic acid residues. Modification was found to proceed appreciably faster than the accompanying loss of enzymic activity. The kinetics of the modification were fitted best by supposing that the two lipoyl groups react with the maleimide at different rates, one being modified at approximately 3.5 times the rate of the other. The loss of complex activity took place at a rate approximately equal to that calculated for the modification of the more slowly reacting lipoic acid residue. The simplest interpretation of this result is that only this residue is essential in the overall catalytic mechanism, but an alternative explanation in which one lipoic acid residue can take over the function of another was not ruled out. The kinetics of inactivation could not be reconciled with an obligatory serial interaction between the two lipoic acid residues. Similar experiments with the fluorescent N-[p-(benzimidazol-2-yl)phenyl]maleimide supported these conclusions, although the modification was found to be less specific than with N-ethylmaleimide. The more rapidly modified lipoic acid residue may be involved in the system of intramolecular transacetylation reactions that couple active sites in the lipoate acetyltransferase component.     

The inactivation of plasma alpha 1-proteinase inhibitor by nitrous acid

Exposure of alpha 1-PI to nitrous acid resulted in a complete inactivation of either of its elastase or trypsin inhibitors activities. Amino acid analyses of the nitrous acid treated inhibitor revealed only losses of one tryphanyl and three lysyl residues. Reductive methylation of alpha 1-PI offered no protection against loss of activity by nitrous acid. Since no further loss of lysyl residues was observed upon exposure of fully active reductively methylated alpha 1-PI to nitrous acid, modification of one tryptophanyl residue appears to be responsible for the inhibitor's sensitivity to nitrous acid. Absorption spectral studies of the nitrous acid treated alpha 1-PI indicated that the tryptophanyl residue was modified to its N-nitroso derivative.     

神经节苷脂联合α-硫辛酸治疗糖尿病神经源性膀胱临床观察

目的：观察α-硫辛酸、神经节苷脂联合治疗糖尿病神经源性膀胱的临床疗效。方法：60例2型糖尿病神经源性膀胱患者,随机分为α-硫辛酸治疗对照组、神经节苷脂治疗对照组,联合治疗组,疗程30d,观察症状体征及治疗前后B超膀胱残余尿量症状的变化,比较不同方案的疗效。结果：α-硫辛酸治疗对照组有效率90％,神经节苷脂治疗对照组有效率90％,联合治疗组有效率95％两组间无统计学差异（P〉0．05）,但3组的治愈率分别为90．0％、50．0％、60．0%,治疗组的治愈率高于两个对照组,有统计学差异（P〈0．05）。结论：α-硫辛酸和神经节苷脂联合治疗糖尿病神经源性膀胱有较好的临床疗效,值得临床推广。     

用荧光技术检测蔬菜中的残留农药

用荧光光谱仪测试了几种用于蔬菜的常用农药的荧光光谱。从一些市售农药上记录到了荧光特征峰,从而可以区分这些农药。据此,可利用荧光光谱仪根据荧光光谱的不同,实时实地和快速地识别这些农药及其在粮食、蔬菜、水果表面上的可能残留。     

Monomeric Creatine Kinase Aggregation and Sodium Dodecyl Sulfate-cyclodextrin Assisted Refolding

Abstract

The monomelic state of creatine kinase (CK) was stably captured at the equilibrium state by employing cysteine residue modifications in the presence of a dénaturant, and at a partially folded state. The partially folded monomeric CK (PF-CK) was aggregated with kinetic order, which was mainly caused by the hydrophobic surface interactions between the CK subunits. The artificial chaperone, described as a SDS-cyclodextrin, was applied to prevent aggregation as well as to refold the PF-CK: SDS treatment onto the monomeric CK can significantly block aggregation and can be successfully refolded in the solutions containing cyclodextrins and DTT. Three types of cyclodextrins such as α-, β-, and γ-cyclodextrins were applied to strip SDS from the enzyme molecule, and each kinetic course was measured. The intrinsic fluorescence changes showed that reactivation occurred and this accompanied the conformational changes. The size exclusion chromatography detected the variously trapped monomeric CKs such as the thiol residue modified PF-CK, the SDS- binding PF-CK, the cyclodextrin treated PF-CK, and the DTT treated SDS-binding PF-CK. Our study demonstrated monomer CK aggregation for the first time; we also demonstrated the complex reassociation of CK during refolding with the aid of the SDS-cyclodextrin, and these pathways followed first-order kinetics.     

合理施用沼渣对土壤钾素有效性影响

通过室内干湿交替培养研究了沼渣对沈阳地区耕地棕壤和草甸土中速效钾含量及其对外源钾固定的影响,结果发现(1)加入沼渣可以明显促进供试土壤中钾素的释放,棕壤和草甸土对照中速效钾含量分别在高于99.24和83.23mg·kg^-1时,干燥导致速效钾含量降低,含6%沼渣的棕壤和草甸土速效钾含量低于130.53和139.29mg·kg^-1时,干燥就会引起层间钾的释放;(2)加入沼渣可以减少土壤对外源钾的固定量,含2%和6%沼渣的棕壤在干湿交替培养15天后,固钾量分别比对照减少了12.51和54.81mg·kg^-1,相应的草甸土分别比对照减少固钾量达62.57和137.66mg·kg^-1,固钾率分别降低了12.93%和28.46%。     

Anion-proton cotransport through the human red blood cell band 3 protein. Role of glutamate 681.

The band 3 protein of the human red blood cell membrane contains a glutamate residue that must be protonated in order for divalent (SO4=) anion transport to take place at an appreciable rate. The carboxyl side chain on this glutamate residue can be converted to the primary alcohol by treatment of intact cells with Woodward's reagent K (N-ethyl-5-phenylisoxazolium 3'-sulfonate) followed by reductive cleavage with BH4-. Edman degradation of CNBr fragments from band 3 labeled in intact cells with Woodward's reagent K and [3H]BH4- showed that Glu681 is heavily labeled under conditions in which Cl- exchange is inhibited, SO4= exchange is accelerated, and Cl- conductance is accelerated. No other glutamate residue in band 3 is detectably labeled under the conditions of these experiments, as demonstrated either by Edman degradation or by the lack of label in major known proteolytic fragments. It is concluded that Glu681 is the binding site for the H+ that is transported with SO4= during band 3-catalyzed H+/SO4= cotransport. This residue is conserved among all species of red cell band 3 (AE1) as well as the related proteins AE2 and AE3. Glu681 is the first amino acid residue in band 3 which has been identified as a binding site for a transported substrate (H+). The functional characteristics of this residue suggest that it lies within the transport pathway and can be alternately exposed to the intracellular and extracellular media.     

Automatic prediction of catalytic residues by modeling residue structural neighborhood

Background  

Prediction of catalytic residues is a major step in characterizing the function of enzymes. In its simpler formulation, the problem can be cast into a binary classification task at the residue level, by predicting whether the residue is directly involved in the catalytic process. The task is quite hard also when structural information is available, due to the rather wide range of roles a functional residue can play and to the large imbalance between the number of catalytic and non-catalytic residues.     

Proteomics for the detection of indirect markers of steroids treatment in bovine muscle

Despite the ban by the European Union, anabolic steroids might still be illicitly employed in bovine meat production. The surveillance of misuse of such potentially harmful molecules is necessary to guarantee consumers’ health. Analytical methods for drug residue control are based on LC‐MS/MS, but their efficacy can be hindered due to undetectable residual concentrations as a result of low‐dosage treatments. Screening methods based on the recognition of indirect biological effects of growth promoters’ administration, such as the alteration of protein expression, can improve the efficacy of surveillance. The present study was aimed at identifying modifications in the muscle protein expression pattern between bulls treated with an ear implant (Revalor‐XS®) containing trenbolone acetate (200 mg) and estradiol (40 mg), and untreated animals. The analysis of skeletal muscle was carried out using a tandem mass tags shotgun proteomics approach. We defined 28 candidate protein markers with a significantly altered expression induced by steroids administration. A subset of 18 candidate markers was validated by SRM and allowed to build a predictive model based on partial least square discriminant analysis. Our findings confirm the effectiveness of the proteomics approach as potential tool to overcome analytical limitations of drug residue monitoring.