Phosphatidylethanolamine synthesis by castor bean endosperm : membrane bilayer distribution of phosphatidylethanolamine synthesized by the ethanolaminephosphotransferase and ethanolamine exchange reactions

A base exchange reaction for synthesis of phosphatidylethanolamine by the endoplasmic reticulum of castor bean (Ricinus comminus L. var Hale) endosperm has been examined. The calculated Michaelis-Menten constant of the enzyme for ethanolamine was 5 micromolar and the optimal pH was 7.8 in the presence of 2 millimolar CaCl₂. l-Serine, N-methylethanolamine and N,N-dimethylethanolamine all reduced ethanolamine incorporation, while d-serine and myo-inositol had little effect. These inhibitions of ethanolamine incorporation were found to be noncompetitive and ethanolamine also noncompetitively inhibited l-serine incorporation by exchange. The activity of the ethanolamine base exchange enzyme was affected by several detergents, with the best activity being obtained with the zwitterionic defjtergent 3-3-cholamidopropyl) dimethylammonio-2-hydroxyl-1-propanesulfonate.     

The hidden dynamics of low coral cover communities

Our study presents a low-cost method (no expensive hardware platforms required) of quantified biomonitoring based on computer image analysis. The negative influence of toxins on surface waters was analysed. The method was verified on widespread freshwater macrophyte Lemna minor to test populations treated with non-ionic detergents. We showed that the proposed automated bioassay has a broad applicability in assessing the negative impacts of aquatic toxicants. This approach enabled fast and precise evaluation of the morphometric parameters of the duckweed test population. We observed that growth rate of L. minor reacts to non-ionic detergents, which is reflected by the change in the surface area. The decrease in the growth of L. minor was revealed at high doses of detergents. This test proved to be highly useful, because it is well repeatable and fast in its implementation. Unlike classical bioassays, the proposed test allows the elimination of measurement errors, resulting from observers’ subjectivity.     

Stimulatory effects of lung cytochrome b5 on benzphetamine N-demethylation in a reconstituted system containing lung cytochrome P450 LgM2

• 1.1. Cytochrome b5 was partially purified from sheep lung microsomes in the presence of detergents Emuigen 913 and cholate by three consecutive DEAE-cellulose and Sephadex G-100 gel filtration chromatographies.
• 2.2. The specific content ofcytochrome b5 was 16.5 nmol/mg protein and purified cytochrome b5 fractions were free of cytochrome P450, NADPH-cytochrome P450 reductase and NADH-cytochrome b5 reductase activities.
• 3.3. The influences of increasing concentrations of lung cytochrome b5 on benzphetamine N-demethylation reactions were examined in four different reconstitution systems containing lung cytochrome P 450 LgM2, lung cytochrome P450 reductase and lipid. In each system concentration of reductase was doubled with respect to former system.
• 4.4. In all systems cytochrome b 5 stimulated benzphetamine Ndemethylase activity especially when cytochrome b5 was present at 0.5:1 molar ratio with respect to cytochrome /P450 LgM2.
• 5.5. Besides, the greatest fold of increase in benzphetamine N-demethylation activity due to addition of cytochrome b5 was observed in System 1 with the lowest concentration of reductase.

     

Enzymic hydrolysis of sphingolipids: Hydrolysis of ceramide glucoside by an enzyme from ox brain

Ceramide glucoside (1-O-glucosido-2-N-acyl-sphingosine) was hydrolysed to ceramide (N-acyl-sphingosine) and glucose by β-glucosidase from ox brain. The reaction was stimulated by the non-ionic detergent, Triton X-100, or by the anionic detergents, cholate or taurocholate. It was not reversible, had optimum pH5·0 (with acetate buffer) or 5·6 (with pyridine buffer), had K_m 1·8×10⁻⁴m and was inhibited by δ-gluconolactone and sphingosine, but not by ceramide or palmitic acid.     

Persistent cAMP binding by the chemotactic cAMP receptor in the presence of a detergent in Dictyostelium discoideum

We have investigated the effect of a number of detergents on the chemotactic cAMP receptor of Dictyostelium discoideum. 13 detergents were tested; cAMP binding was well preserved only in the presence CHAPS (3[3-cholamidopropyl)dimethylammonio]-1-propanesulphonate) and Zwittergent 3–8 (N-octyl-N,N-dimethyl-3-ammonio-1-propanesulphonate). In the presence of Zwittergent 3–8, cAMP bound to the receptor rapidly exchanged with free cAMP. In contrast, cAMP was persistently bound to the receptor following the addition of CHAPS to membrane-bound receptors pre-equilibrated with cAMP. Binding isotherms indicated that all cAMP-binding sites were similarly affected by CHAPS. The cyclic nucleotide binding specificity of the binding sites that became persistently occupied by cAMP was identical to that of the chemotactic cAMP receptor. Cyclic AMP was not chemically modified by persistent binding. The non-exchanging cAMP-receptor complex was insensitive to modulation by guanine nucleotides and salts such as CaCl₂, MgCl₂, potassium phosphate and ammonium sulphate. We conclude that CHAPS freezes the cAMP-receptor, blocking exchange of free ligand with empty or occupied cAMP-binding sites.     

Induction of the blue form of bacteriohodopsin by low concentrations of sodium dodecyl sulfate

The effects produced on bacteriorhodopsin by low concentrations of several detergents have been studied by absorption and fourth-derivative spectrophotometry. Sodium dodecyl sulfate induces the appearance of the blue form of bacteriorhodopsin (λ_max = 600 nm) at pH values up to 7.0 in a reversible manner. The apparent pK of the purple-to-blue transition raised with increasing concentration of SDS. Of the other detergents tested, only sodium dodecyl-N-sarcosinate showed a slight red-shift of the absorption band to 580 nm, whereas sodium taurocholate, Triton X-100 and cetyltrimethylammonium bromide did not favour the appearance of the blue form. The effect of SDS was found to be consistent with a localized conformational change that moves away the counter-ion of the protonated Schiff base.     

Two-dimensional electrophoresis of membrane proteins factors affecting resolution of rat-liver microsomal proteins

A comparison has been made of published techniques for the resolution of rat liver microsomal proteins by two-dimensional electrophoresis. The method of Kaderbhai and Freedman (Biochim. Biophys. Acta 601 (1980) 21-20) gives good resolution of acidic proteins but excludes hydrophobic integral membrane proteins of pI > 7, including cytochrome P-450 apoproteins. The method of Vlasuk and Walz (Anal. Biochem. 105 (1980) 112–120) gives good resolution of proetins of pI 5–8, including cytochromes P-450, but fails to resolve a major acidic protein of pI < 5. Isoelectric focusing of microsomal proteins is improved by the use of high concentrations of urea and low concentrations of sample proteins. Zwitterionic detergents of the general formula R·N⁺(CH₃)₂·CH₂CH₂CH₂SO₃^? are effective in solubilizing microsomal proteins, either alone or in presence of non-ionic detergent; compounds with a long alkyl chain (C₁₄ or C₁₆) are most effective. Isoelectric focusing of microsomal proteins solubilized by zwitterionic detergents did not give good resolution, probably because of incomplete dissociation and denaturation of the proteins. These detergents could not be used in the presence of high concentrations of urea. Although no single method of two-dimensional electrophoresis gives complete resolution of the whole range of microsomal proteins, conditions can be optimized for specific sets of proteins of interest. The technique can be used to monitor differences in microsomal composition between rat strains, or following induction, and for a variety of other studies.     

Synthesis of metal complexes of peptide-phosphinites: Metal complexes in asymmetric Heck reaction

The synthesis of seven peptide-derived phosphinites, N-Boc-Phe-Tyr(OPPh₂)-OMe (4), N-Boc-Phe-Tyr(OPEt₂)-OMe (5), N-Boc-Phe-Tyr(OPCy₂)-OMe (6), N-Boc-Phe-Ser(OPPh₂)-OMe (7), N-Boc-Phe-Ser(OP^tBu₂)-OMe (8), N-Boc-Phe-Thr(OPPh₂)-OMe (9), N-Boc-Phe-Thr(OP^tBu₂)-OMe (10) is reported. These ligands are readily coordinated to Pd(II) and Pt(II) centers giving the corresponding complexes of the type ML₂Cl₂ (11-20). The palladium complexes, [N-Boc-Phe-Tyr(OPPh₂)-OMe]₂PdCl₂ (16), [N-Boc-Phe-Tyr(OPEt₂)-OMe]₂PdCl₂ (17), [N-Boc-Phe-Tyr(OPCy₂)-OMe]₂PdCl₂ (18), [N-Boc-Phe-Ser(OPPh₂)-OMe]₂PdCl₂ (19) and [N-Boc-Phe-Thr(OPPh₂)-OMe]₂PdCl₂ (20) catalyze the asymmetric phenylation of 2,3-dihydrofuran in moderate to high yields with high ee’s. The steric and electronic influences of the ligand substituents in driving the catalytic process are also discussed.     

A gas chromatographic method for quantification of detergents frequently used in membrane protein structural studies

A novel and reliable gas chromatography-flame ionization detection (GC-FID) method that can separate and quantify detergents frequently used in membrane protein structural studies has been developed. Different detergents were identified through FID peaks with different retention times. A quadratic regression curve was found to fit the integrated FID peak area against different detergent concentrations. Detergents can be quantified as low as the nanogram level: lauryl-dimethylamine-N-oxide (LDAO), 5 ng; dodecyl maltoside (DDM), 10 ng; and dodecyl phosphocholine (DPC), 50 ng. This method can be applied directly to measure detergent concentration and molar ratio of membrane protein to detergents during membrane protein extraction, purification, concentration, and crystallization.     

Synthesis of Fucosyl-N-Acetylglucosamine Disaccharides by Transfucosylation Using α-l-Fucosidases from Lactobacillus casei

AlfB and AlfC α-l-fucosidases from Lactobacillus casei were used in transglycosylation reactions, and they showed high efficiency in synthesizing fucosyldisaccharides. AlfB and AlfC activities exclusively produced fucosyl-α-1,3-N-acetylglucosamine and fucosyl-α-1,6-N-acetylglucosamine, respectively. The reaction kinetics showed that AlfB can convert 23% p-nitrophenyl-α-l-fucopyranoside into fucosyl-α-1,3-N-acetylglucosamine and AlfC at up to 56% into fucosyl-α-1,6-N-acetylglucosamine.     

Characterization and Two-Dimensional Crystallization of Membrane Component AlkB of the Medium-Chain Alkane Hydroxylase System from Pseudomonas putida GPo1

The alkane hydroxylase system of Pseudomonas putida GPo1 allows it to use alkanes as the sole source of carbon and energy. Bacterial alkane hydroxylases have tremendous potential as biocatalysts for the stereo- and regioselective transformation of a wide range of chemically inert unreactive alkanes into valuable reactive chemical precursors. We have produced and characterized the first 2-dimensional crystals of the integral membrane component of the P. putida alkane hydroxylase system, the nonheme di-iron alkane monooxygenase AlkB. Our analysis reveals for the first time that AlkB reconstituted into a lipid bilayer forms trimers. Addition of detergents that do not disrupt the AlkB oligomeric state (decyl maltose neopentyl glycol [DMNG], lauryl maltose neopentyl glycol [LMNG], and octaethylene glycol monododecyl ether [C₁₂E₈]) preserved its activity at a level close to that of the detergent-free control sample. In contrast, the monomeric form of AlkB produced by purification in n-decyl-β-d-maltopyranoside (DM), n-dodecyl-β-d-maltopyranoside (DDM), octyl glucose neopentyl glycol (OGNG), and n-dodecyl-N,N-dimethylamine-N-oxide (LDAO) was largely inactive. This is the first indication that the physiologically active form of membrane-embedded AlkB may be a multimer. We present for the first time experimental evidence that 1-octyne acts as a mechanism-based inhibitor of AlkB. Therefore, despite the lack of any significant full-length sequence similarity with members of other monooxygenase classes that catalyze the terminal oxidation of alkanes, AlkB is likely to share a similar catalytic mechanism.     

Fine-scale differences in genetic and census population size ratios between two stream fishes

Comparing the ratio of effective number of breeders (N _b ) to adult population size (N) among closely related coexisting species can provide insights into the role of life history on N _b /N ratios and inform conservation programs towards limiting the loss of evolutionary potential in natural populations. We estimated N _b and N in two coexisting salmonid fishes (Brook trout and Atlantic salmon) for 3–4 consecutive years in two small, adjacent streams in Newfoundland, Canada, using mark-recapture (N), linkage disequilibrium (N _b(LD)), and sibship frequency approaches (N _b(Sib) ). We found that N _b /N ratios were about 20-fold greater in Atlantic salmon than in brook trout (mean 0.20, range 0.06–0.56 vs. mean 0.02, range 0.01–0.05, respectively). This difference was consistent across N _b estimators. In addition, we found that removing migrants reduced N _b : the strength of the effect was weak for N _b(LD) and much stronger for N _b(Sib). Our results highlight the importance of subtle ecological differences and gene flow in shaping N _b /N. They also provide some evidence that the linkage between demographic and evolutionary processes varies between closely related taxa and suggest that a more complete understanding of the N _b /N range across various species is an important component of conservation genetics and management.     

The ammonia-catalyzed release of glycoprotein <Emphasis Type="Italic">N</Emphasis>-glycans

Despite the great significance of release and analysis of glycans from glycoproteins, the existing N-glycan release methods are undermined by some limitations and deficiencies. The traditional enzymatic protocols feature high N-glycan release specificity but are generally costly and inefficient for some types of N-glycans. The existing chemical methods require harsh reaction conditions or are accompanied by the remarkable formation of by-products. Herein, we describe a versatile chemical method for the release and analysis of N-glycans from glycoproteins. This method differs from the existing methods as only aqueous ammonia is used to catalyze the N-glycan release reactions. Optimization of reaction conditions was performed using RNase B as a model glycoprotein and the obtained results indicated a highest N-glycan yield in ammonia at 60 °C for 16 h. Comparison of this method with traditional enzymatic protocols and recently reported NaClO methods confirmed the good reliability and efficiency of the novel approach. We also successfully applied this method to some complex biological samples, such as Ginkgo seed protein, fetal bovine serum (FBS) and hen egg white, and demonstrated its great compatibility with various neutral N-glycans, core α-1,3-fucosylated N-glycans and sialylated N-glycans. This method is very simple and cost-effective, enabling convenient analysis and large-scale preparation of released reducing N-glycans from various biological samples for structural and functional glycomics studies.     

Partitioning of Triton X-100, deoxycholate and C10EO8 into bilayers composed of native and hydrogenated egg yolk sphingomyelin

We have used isothermal titration calorimetry (ITC) to study the thermodynamics of Triton X-100 (T_X-100), deoxycholate and decyl octaethylene glycol (C₁₀EO₈) penetration into bilayers composed of native (ESM) and hydrogenated egg yolk sphingomyelin (DHSM). Light scattering measurements were used to study the point of saturation (R_e,sat) and the onset of solubilization of membranes by the detergents. We found that DHSM bilayers at 25 °C were much more resistant to detergent partitioning (lower K) and gave higher reaction enthalpies (ΔH) for all three detergents compared to the ESM bilayer system. Because DHSM lacks double bonds (Δ^4trans and some cis bonds as well), attractive acyl chain interactions are favored in membranes of this lipid class. The high stability and cohesion of DHSM in membranes could be a crucial functional property of this lipid as it is enriched in eye lens membranes.     

Exposure to nitrogen does not eliminate N2 fixation in the feather moss Pleurozium schreberi (Brid.) Mitt.

Background and aims

The feather moss Pleurozium schreberi (Brid.) Mitt. is colonized by cyanobacteria, which fix substantial amounts of atmospheric nitrogen (N) in pristine and N-poor ecosystems. Cyanobacterial N₂ fixation is inhibited by N deposition. However, the threshold of N input that leads to the inhibition of N₂ fixation has not been adequately investigated. Further, the ability of N₂ fixation to recover in mosses from high N deposition areas has not been studied to date.

Methods

We conducted two laboratory studies in which we (1) applied a range of concentrations of N as NH₄NO₃ to mosses from low N-deposition areas, and (2) we deprived mosses from a high N-deposition area of N to test their ability to recover N₂ fixation.

Results

Higher addition rates (up to 10 kg N ha^?1) did not systematically inhibit N₂ fixation in P. schreberi. Conversely, upon weeks of N deprivation of mosses from a high N environment, N₂ fixation rates increased.

Conclusions

The threshold of total N deposition above which N₂ fixation in P. schreberi is inhibited is likely to be > 10 kg N ha^?1. Further, cyanobacteria are able to recover from high N inputs and are able to fix atmospheric N₂ after a period of N deprivation.     

Synthesis of new N-substituted 3,4,5-trihydroxypiperidin-2-ones from d-ribono-1,4-lactone

d-Ribono-1,4-lactone was treated with ethylamine in DMF to afford N-ethyl-d-ribonamide 8a in quantitative yield. Using this reaction procedure, N-butyl, N-hexyl, N-dodecyl, N-benzyl, N-(3-methyl-pyridinyl)-, N-(2-hydroxy-ethyl)-, and N-(2-cyano-ethyl)-d-ribonamides 8b-h were obtained in quantitative yield. Bromination of the amides 8a-e with acetyl bromide in dioxane followed by acetylation gave 2,3,4-tri-O-acetyl-5-bromo-5-deoxy-N-ethyl, N-butyl, N-hexyl, N-dodecyl, and N-benzyl-d-ribonamides 9a-e in 40-54% yields. To obtain 2,3,4-tri-O-acetyl-5-bromo-5-deoxy-N-(3-methyl-pyridinyl)-, N-(2-hydroxy-ethyl)-, and N-(2-cyano-ethyl)-9f-h, the bromination is necessary before the amidation reaction. Treatment of the bromoamides 9a-h with NaH in DMF followed by methanolysis affords N-alkyl-d-ribono-1,5-lactams 12a-h in quantitative yield.     

<Emphasis Type="Italic">N</Emphasis>-Glycosylation of an IgG antibody secreted by <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> BY-2 cells can be modulated through co-expression of human β-1,4-galactosyltransferase

Nicotiana tabacum BY-2 suspension cells have several advantages that make them suitable for the production of full-size monoclonal antibodies which can be purified directly from the culture medium. Carbohydrate characterization of an antibody (Lo-BM2) expressed in N. tabacum BY-2 cells showed that the purified Lo-BM2 displays N-glycan homogeneity with a high proportion (>70%) of the complex GnGnXF glycoform. The stable co-expression of a human β-1,4-galactosyltransferase targeted to different Golgi sub-compartments altered Lo-BM2N-glycosylation and resulted in the production of an antibody that exhibited either hybrid structures containing a low abundance of the plant epitopes (α-1,3-fucose and β-1,2-xylose), or a large amount of galactose-extended N-glycan structures. These results demonstrate the suitability of stable N-glycoengineered N. tabacum BY-2 cell lines for the production of human-like antibodies.     

An acidic,thermostable exochitinase with β-<Emphasis Type="Italic">N</Emphasis>-acetylglucosaminidase activity from <Emphasis Type="Italic">Paenibacillus barengoltzii</Emphasis> converting chitin to <Emphasis Type="Italic">N</Emphasis>-acetyl glucosamine

Background

N-acetyl-β-D-glucosamine (GlcNAc) is widely used as a valuable pharmacological agent and a functional food additive. The traditional chemical process for GlcNAc production has some problems such as high production cost, low yield, and acidic pollution. Hence, to identify a novel chitinase that is suitable for bioconversion of chitin to GlcNAc is of great value.

Results

A novel chitinase gene (PbChi74) from Paenibacillus barengoltzii was cloned and heterologously expressed in Escherichia coli as an intracellular soluble protein. The gene has an open reading frame (ORF) of 2,163 bp encoding 720 amino acids. The recombinant chitinase (PbChi74) was purified to apparent homogeneity with a purification fold of 2.2 and a recovery yield of 57.9%. The molecular mass of the purified enzyme was estimated to be 74.6 kDa and 74.3 kDa by SDS-PAGE and gel filtration, respectively. PbChi74 displayed an acidic pH optimum of 4.5 and a temperature optimum of 65°C. The enzyme showed high activity toward colloidal chitin, glycol chitin, N-acetyl chitooligosaccharides, and p-nitrophenyl N-acetyl β-glucosaminide. PbChi74 hydrolyzed colloidal chitin to yield N- acetyl chitobiose [(GlcNAc)₂] at the initial stage, which was further converted to its monomer N-acetyl glucosamine (GlcNAc), suggesting that it is an exochitinase with β-N-acetylglucosaminidase activity. The purified PbChi74 coupled with RmNAG (β-N-acetylglucosaminidase from Rhizomucor miehei) was used to convert colloidal chitin to GlcNAc, and GlcNAc was the sole end product at a concentration of 27.8 mg mL^-1 with a conversion yield of 92.6%. These results suggest that PbChi74 may have great potential in chitin conversion.

Conclusions

The excellent thermostability and hydrolytic properties may give the exochitinase great potential in GlcNAc production from chitin. This is the first report on an exochitinase with N-acetyl-β-D-glucosaminidase activity from Paenibacillus species.

     

Comparative theoretical studies of differently bridged nitramino-substituted ditetrazole 2-<Emphasis Type="Italic">N</Emphasis>-oxides with high detonation performance and an oxygen balance of around zero

In this work, six (A–F) nitramino (–NHNO₂)-substituted ditetrazole 2-N-oxides with different bridging groups (–CH₂–, –CH₂–CH₂–, –NH–, –N=N–, and –NH–NH–) were designed. The six compounds were based on the parent compound tetrazole 2-N-oxide, which possesses a high oxygen balance and high density. The structure, heat of formation, density, detonation properties (detonation velocity D and detonation pressure P), and the sensitivity of each compound was investigated systematically via density functional theory, by studying the electrostatic potential, and using molecular mechanics. The results showed that compounds A–F all have outstanding energetic properties (D: 9.1–10.0 km/s; P: 38.0–46.7 GPa) and acceptable sensitivities (h ₅₀: 28–37 cm). The bridging group present was found to greatly affect the detonation performance of each ditetrazole 2-N-oxide, and the compound with the –NH–NH– bridging group yielded the best results. Indeed, this compound (F) was calculated to have comparable sensitivity to the famous and widely used high explosive 1,3,5,7-tetranitro-1,3,5,7-tetrazocane (HMX), but with values of D and P that were about 8.7% and 19.4% higher than those for HMX, respectively. The present study shows that tetrazole 2-N-oxide is a useful parent compound which could potentially be used in the design of new and improved high-energy compounds to replace existing energetic compounds such as HMX.     

Cultivar variation in cotton photosynthetic performance under different temperature regimes

Cotton (Gossypium hirsutum L.) yields are impacted by overall photosynthetic production. Factors that influence crop photosynthesis are the plants genetic makeup and the environmental conditions. This study investigated cultivar variation in photosynthesis in the field conditions under both ambient and higher temperature. Six diverse cotton cultivars were grown in the field at Stoneville, MS under both an ambient and a high temperature regime during the 2006–2008 growing seasons. Mid-season leaf net photosynthetic rates (P_N) and dark-adapted chlorophyll fluorescence variable to maximal ratios (F_v/F_m) were determined on two leaves per plot. Temperature regimes did not have a significant effect on either P_N or F_v/F_m. In 2006, however, there was a significant cultivar × temperature interaction for P_N caused by PeeDee 3 having a lower P_N under the high temperature regime. Other cultivars’ P_N were not affected by temperature. FM 800BR cultivar consistently had a higher P_N across the years of the study. Despite demonstrating a higher leaf F_v/F_m, ST 5599BR exhibited a lower P_N than the other cultivars. Although genetic variability was detected in photosynthesis and heat tolerance, the differences found were probably too small and inconsistent to be useful for a breeding program.