A comparison of the poly(hexamethylenebiguanidinium chloride) assay and a neutral equivalent method for the determination of alginates in industrial liquors extracted from brown seaweed

The poly(hexamethylenebiguanidinium chloride) assay and a neutral equivalent method have been used to estimate the sodium alginate content of industrial liquors extracted from brown seaweed. With the liquor samples examined, agreement to within 5% was achieved with the two methods under defined alginate concentration conditions. Statistical evaluation of the data using a paired comparison t-test has shown that a difference of only 0·10±0·05 can be expected with 95% confidence, between the two methods in the analysis of liquor samples with sodium alginate contents in the range 2·00–3·83 mg ml⁻¹. In the majority of cases the PHMBH⁺ Cl⁻ method gave a slightly higher estimate that the neutral equivalent method. Consideration of the practical aspects of the two alternative methods allows this difference to be explained. The simplicity of the PHMBH⁺ Cl⁻ assay renders it suitable for the rapid screening of large numbers of alginate samples.     

Use of the mitochondria toxicity assay for quantifying the viable cell density of microencapsulated jurkat cells

The mitochondria toxicity assay (MTT assay) is an established method for monitoring cell viability based on mitochondrial activity. Here the MTT assay is proposed for the in situ quantification of the living cell density of microencapsulated Jurkat cells. Three systems were used to encapsulate the cells, namely a membrane consisting of an interpenetrating polyelectrolyte network of sodium cellulose sulphate/poly(diallyldimethylammonium chloride) (NaCS/PDADMAC), a calcium alginate hydrogel covered with poly(L ‐lysine) (Ca‐alg‐PLL), and a novel calcium alginate‐poly(ethylene glycol) hybrid material (Ca‐alg‐PEG). MTT results were correlated to data obtained by the trypan blue exclusion assay after release of the cells from the NaCS/PDADMAC and Ca‐alg‐PLL capsules, while a resazurin‐based assay was used for comparison in case of the Ca‐alg‐PEG material. Analysis by MTT assay allows quick and reliable determination of viable cell densities of encapsulated cells independent of the capsule material. The assay is highly reproducible with inter‐assay relative standard deviations below 10%. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:986–993, 2013     

Cholinesterase assay by gas--solid chromatography.

The determination of serum cholinesterase activity was based on the hydrolysis of butyrylcholine chloride, solvent extraction, and the quantitative estimation of the hydrolytic product, butyric acid, by gas-solid chromatography. Using this procedure, eptimum conditions of pH and buffer strength for the cholinesterase activity were pH 8.0 and 0.05 m Tris-HCl, respectively. The cholinesterase activities in blood samples from healthy subjects, as determined by this method, were in the range of normal values reported in the literature. The effects of eserine and other drugs on the cholinesterase activity were also reported. The high accuracy (1.1%) and high sensitivity which permit the determination of butyric acid at levels down to 1 μg/ml make this procedure attractive as a serum cholinesterase assay.     

Isolation and characterization of an alginate lyase from Klebsiella aerogenes

The bacterium Klebsiella aerogenes (type 25) produced an inducible alginate lyase, whose major activity was located intracellularly during all growth phases. The enzyme was purified from the soluble fraction of sonicated cells by ammonium sulfate precipitation, anion- and cation-exchange chromatography and gel filtration. The apparent molecular weight of purified alginate lyase of 28,000 determined by gel filtration and of 31,600 determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the active enzyme was composed of a single polypeptide. The alginate lyase displayed a pH optimum around 7.0 and a temperature optimum around 37°C. The purified enzyme depolymerized alginate by a lyase reaction in an endo manner releasing products which reacted in the thiobarbituric acid assay and absorbed strongly in the ultraviolet region at 235 nm. The alginate lyase was specific for guluronic acidrich alginate preparations. Propylene glycol esters of alginate and O-acetylated bacterial alginates were poorly degraded by the lyase compared with unmodified polysaccharide. The guluronate-specific lyase activity was applied in an enzymatic method to detect mannuronan C-5 epimerase in three different mucoid (alginate-synthesizing) strains of Pseudomonas aeruginosa. This enzyme which converts polymannuronate to alginate could not be demonstrated either extracellularly or intracellularly in all strains suggesting the absence of a polymannuronate-modifying enzyme in P. aeruginosa.Abbreviations poly(ManA) (1–4)--D-mannuronan - poly(GulA) (1–4)--L-guluronan - TBA 2-thiobarbituric acid     

Synthesis and characterization of a biotin-alginate conjugate and its application in a biosensor construction

Biotin was covalently coupled with alginate in an aqueous-phase reaction by means of carbodiimide-mediated activation chemistry to provide a biotin-alginate conjugate for subsequent use in biosensor applications. The synthetic procedure was optimized with respect to pH of the reaction medium (pH 6.0), the degree of uronic acid activation (20%), and the order of addition of the reagents. The biotin-alginate conjugate was characterized by titration with 2-anilinonaphthalene-6-sulfonic acid (2,6-ANS), 4-hydroxyazobene-2'-carboxylic acid (HABA) and by an HPSEC-MALLS analytical method as well as by FTIR and 13C NMR spectroscopy. As a compromise between the need for a high percent of molar modification of the alginate, on one hand, and sufficient gelling capability, on the other hand, an optimal modification of 10-13% of biotin-alginate was used. The new biotin-alginate conjugate was used for the encapsulation of bioluminescent reporter cells into microspheres. A biosensor was prepared by conjugating these biotinylated alginate microspheres to the surface of a streptavidin-coated optical fiber, and the performance of the biosensor was demonstrated in the determination of the antibiotic, mitomycin C as a model toxin.     

A simple and specific determination of glycine in biological samples

A simple specific assay was developed for the determination of glycine in a solution containing other amino acids. Hippuric acid was obtained after reacting glycine with benzoyl chloride and was extracted with ethyl acetate. It was then reacted with acetic anhydride, p-dimethylaminobenzaldehyde, and pyridine for color development. The amount of glycine (1 to 100 μg) in the original solution could be determined by measuring the absorbance (458 nm) of this chromogen. This procedure was applied on an amino acid mixture, urine, serum, blood, and liver homogenate.     

Pilot plant scale extraction of alginates from Macrocystis pyrifera 3. Precipitation, bleaching and conversion of calcium alginate to alginic acid

Three steps of the alginate production process were studied at pilot plantlevel. The effect of the amount of calcium chloride used during theprecipitation was measured in terms of filtration time of the precipitatedcalcium alginate. Three different proportions of calcium chloride per gramof alginate were tested. The best proportion used was 2.2 parts ofcalcium chloride per one part of alginate, yielding a filtration rate of 97.9L min^-1 on a screen area of 1.32 m². The method ofadding the solutions and the degree of mixing are discussed as other factorsaffecting the precipitation step. The effect of bleaching the calciumalginate with sodium hypochlorite (5%) was studied. Seven proportions,ranging from 0 to 0.77 mL of sodium hypochlorite per gram of sodiumalginate were tested. The effect of hypochlorite was compared foralginates with three different viscosities. Using alginates with mediumviscosity (300–500 mPa s), the best proportion was 0.4 mL hypochloriteper gram of alginate, yielding an alginate of light cream color with 20%less viscosity than the control. Alginates with lower viscosity showed asmaller loss of viscosity. The effect of pH during conversion of calciumalginate to alginic acid was determined using four combinations of pH,ranging from 2.2 to 1.6, in three acid washings. The extent of conversionwas determined by measuring the percent reduction of the alginate viscosity(RV) in 1% solution before and after adding a sequestrant of calcium. When a pH 1.8 or 1.6 was used for each washing, only two washings werenecessary to produce a RV lower than 40% (maximum recommended). The use of pH 2 required three acid washings to produce the same effect. The pH 2.2 did not remove enough calcium, even with three washings,the RV of the resulting sodium alginate being greater that 40%. Theresults of these experiments provide the information that producers needwhen deciding the best parameters to obtain a product with the desiredcharacteristics.     

产双功能褐藻胶裂解酶菌株的筛选与初步研究

目的：双功能褐藻胶裂解酶既能降解聚β-D-甘露糖醛酸,又能降解聚α-L-古罗糖醛酸,可以用一种酶来制备不同结构的褐藻胶寡糖。本文的目的是筛选能产生双功能褐藻胶裂解酶的菌株,对其产酶曲线和降解产物作初步研究。方法：利用唯一碳源培养基筛选产生褐藻胶裂解酶的菌株,通过16SrDNA序列比对进行菌种鉴定,通过在凝胶上检测褐藻胶裂解酶活性来判断发酵上清液中褐藻胶裂解酶的数量及分子量,利用薄层层析确定降解褐藻胶的终产物组成。结果：从褐藻上筛选到一株海洋细菌QY107,鉴定为弧菌属细菌。发酵120h时褐藻胶裂解酶产量为12．32U／mL,其发酵液上清中只含有一种褐藻胶裂解酶,分子量在28kDa左右,并且对聚β—D-甘露糖醛酸和聚α-L-古罗糖醛酸都能降解,降解褐藻胶的终产物主要为三糖。结论：本文筛选到一株弧菌QY107,其发酵液上清中只有一种双功能褐藻胶裂解酶,可用于大量制备褐藻胶三糖。推测该酶具有特殊的催化腔结构,对其结构与功能相互关系的研究可能会发现新的底物结合与催化机制。酶解制备褐藻胶寡糖因其环保高效而越来越受到人们的重视,因此该菌株能促进海洋寡糖类生物制品的开发,在医药、食品、农业、生物燃料等领域具有广阔的应用前景。     

Synthesis and application of fluorescent labeled nucleotides to assay DNA damage

A facile method was developed to covalently attach a fluorophore to the 5'-phosphate of a nucleic acid. The procedure, illustrated by coupling 5'-dNmp (N = A,C,G,T) with 5-dimethylaminonaphthalene 1-sulfonyl chloride, commonly known as Dansyl chloride, involves 5'-phosphoramidation with ethylenediamine (EDA) followed by conjugation of the free aliphatic amino group of the phosphoramidate with Dansyl chloride. This method is also applicable to multi-incorporation of fluorescent labels in the nucleic acids. The reaction of 5'-Amp with a polyamine such as poly L-lysine (PLL, mol. wt., 4000) resulted in a phosphoramidate with multiple amino groups, which after isolation and conjugation with fluorescamine gave dAmp with multilabeled fluorophores. A condition was devised to separate the four dansylated mononucleotides of DNA, conjugated via ethylenediamine linker, by reverse phase HPLC. The elution profile could be monitored with a variable wavelength detector at 254 nm and 340 nm corresponding to the absorption of the nucleotides and the dansyl moiety, respectively. The detection limit was 2 nmol at 254 nm. The use of a fluorescence detector enhanced the detection sensitivity to a sub-picomole level (200 fmol). Samples of a DNA model, d(pCpGpTpA) and calf-thymus DNA were digested enzymatically to 5'-mononucleotides and labeled with Dansyl chloride. HPLC analysis of the dansylated digests from these samples, both before and after irradiation, suggests that the combination of enzymatic digestion and fluorescence postlabeling could be a novel approach to assay DNA damage.     

一种微量、快速测定植物种子b- 淀粉酶活性的方法

针对3, 5-二硝基水杨酸(DNS)法的缺点, 以对硝基苯酚麦芽五糖(PNPG5)为底物, 建立并改进了植物种子b-淀粉酶活性测定的方法。本方法具有微量和快速的特点; 进一步通过对4种植物材料种子的酶活性测定以及a-淀粉酶的干扰评估实验, 证实了该法可以运用于植物种子b-淀粉酶活性的专一性测定。     

Self-cross-linking polyelectrolyte complexes for therapeutic cell encapsulation

Self-cross-linking polyelectrolytes are used to strengthen the surface of calcium alginate beads for cell encapsulation. Poly([2-(methacryloyloxy)ethyl]trimethylammonium chloride), containing 30 mol % 2-aminoethyl methacrylate, and poly(sodium methacrylate), containing 30 mol % 2-(methacryloyloxy)ethyl acetoacetate, were prepared by radical polymerization. Sequential deposition of these polyelectrolytes on calcium alginate films or beads led to a shell consisting of a covalently cross-linked polyelectrolyte complex that resisted osmotic pressure changes as well as challenges with citrate and high ionic strength. Confocal laser fluorescence microscopy revealed that both polyelectrolytes were concentrated in the outer 7-25 microm of the calcium alginate beads. The thickness of this cross-linked shell increased with exposure time. GPC studies of solutions permeating through analogous flat model membranes showed molecular weight cut-offs between 150 and 200 kg/mol for poly(ethylene glycol), suitable for cell encapsulation. C 2C 12 mouse cells were shown to be viable within calcium alginate capsules coated with the new polyelectrolytes, even though some of the capsules showed fibroid overcoats when implanted in mice due to an immune response.     

Disorder–order transitions induced in anionic homopolypeptides by cationic detergents

CD spectra have been obtained for poly(L -glutamic acid) and poly(L -aspartic acid) as functions of temperature and concentration of cationic detergents. Dodecylammonium chloride induces a coil–helix transition in fully ionized poly(L -glutamic acid). The interaction of the monomeric detergent with the polypeptide is responsible for the conformational transition. The detergent concentration required to produce the transition is independent of temperature. The CD of fully ionized poly(L -aspartic acid) is nearly unaffected by dodecylammonium chloride, in marked contrast to the situation found with poly(L -glutamic acid). However, these results do not imply that dodecylammonium chloride interacts differently with aspartyl and glutamyl residues. The observed results can be accounted for by the well-known fact that the glutamyl residue has a higher helix-forming tendency that the aspartyl residue. Cetyltrimethylammonium chloride destabilizes the helical form of poly(L -glutamic acid). This detergent presents an exception to the usual ability of ionic detergents to promote formation of ordered structures in oppositely charged homopolypeptides.     

Rapid and simultaneous high-performance liquid chromatography assay of polyamines and monoacetylpolyamines in biological specimens

A rapid, resolutive and reproducible reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed for polyamines and acetylpolyamines by adopting pre-column derivatization with benzoyl chloride. In a single run lasting less than 15 min ten polyamines were separated as well as traces of benzoic acid, methylbenzoate and benzoic anhydride. These contaminants, produced during the derivatization reaction, were almost all eliminated by washing steps envisaged in the same procedure. This simple and sensitive method can be applied to routine determination of polyamines in biological samples. A fine application of this procedure to the determination of endogenous content of polyamines in chick embryo retina was reported.     

A rapid,sensitive, simple plate assay for detection of microbial alginate lyase activity

Screening of microorganisms capable of producing alginate lyase enzyme is commonly carried out by investigating their abilities to grow on alginate-containing solid media plates and occurrence of a clearance zone after flooding the plates with agents such as 10% (w/v) cetyl pyridinium chloride (CPC), which can form complexes with alginate. Although the CPC method is good, advantageous, and routinely used, the agar in the media interferes with the action of CPC, which makes judgment about clearance zones very difficult. In addition, this method takes a minimum of 30 min to obtain the zone of hydrolysis after flooding and the hydrolyzed area is not sharply discernible. An improved plate assay is reported herein for the detection of extracellular alginate lyase production by microorganisms. In this method, alginate-containing agar plates are flooded with Gram's iodine instead of CPC. Gram's iodine forms a bluish black complex with alginate but not with hydrolyzed alginate, giving sharp, distinct zones around the alginate lyase producing microbial colonies within 2–3 min. Gram's iodine method was found to be more effective than the CPC method in terms of visualization and measurement of zone size. The alginate-lyase-activity area indicated using the Gram's iodine method was found to be larger than that indicated by the CPC method. Both methods (CPC and Gram's iodine) showed the largest alginate lyase activity area for Saccharophagus degradans (ATCC 43961) followed by Microbulbifer mangrovi (KCTC 23483), Bacillus cereus (KF801505) and Paracoccus sp. LL1 (KP288668) grown on minimal sea salt medium. The rate of growth and metabolite production in alginate-containing minimal sea salt liquid medium, followed trends similar to that of the zone activity areas for the four bacteria under study. These results suggested that the assay developed in this study of Gram's iodine could be useful to predict the potential of microorganisms to produce alginate lyase. The method also worked well for screening and identification of alginate lyase producers and non-producers from environmental samples on common laboratory media. They did this by clearly showing the presence or absence of clearance zones around the microbial colonies grown. This new method is rapid, efficient, and could easily be performed for screening a large number of microbial cultures. This is the first report on the use of Gram's iodine for the detection of alginate lyase production by microorganisms using plate assay.     

酵母全细胞苯丙氨酸鲜氨酶（PAL）活性的紫外分光光度测定法

本文报告以L-苯丙氨酸 (L-phe) 为底物,酵母全细胞作酶源,酶促生成产物反式-肉桂酸 (t-Ca)测定苯丙氨解氨酸 (PAP,EC_(4.3.1.5) 活性的紫外分光光度法。测定程序包括标准物质t-Ca的加样试验,绝对回收率试验,线性回归分析的整套定量分析研宄步骤,建立了一套经过修改的Kalghatgi和Subba Rao(1975) PAL 活性测定法。此法具有良好的准确度和精密度,已经用于评价具有PAL活性的酵母菌株在液体培养物中细胞生长和PAL活性形成的时间过程研究。     

A procedure for total protein determination with special application to allergenic extract standardization

A method for total protein determination of allergenic extracts has been developed and evaluated. Samples were hydrolyzed with 5 M NaOH followed by colorimetric determination with ninhydrin of the released amino acids using bovine serum albumin as the standard. The entire procedure was carried out in disposable plastic tubes. Substances (glycerol, phenol and mannitol) commonly present in allergenic extracts manufactured for human use did not affect the assay results. Analyses of four different pollen extracts by the method gave good agreement with amino acid analyses. Other methods of analysis (total N, protein N unit assay, Lowry) gave more variable results compared with amino acid analysis. Analysis of the total protein content of 53 different lots of allergenic extracts gave narrow ranges of values for each species. Standardized mite extracts analyzed for total protein by US FDA-licensed manufacturers using this assay showed a good correlation of biological activity with total protein.     

Simple high-performance liquid chromatographic method to verify the direct barbituric acid assay for urinary cotinine

An isocratic HPLC method is described to determine urinary concentrations of nicotine and cotinine after derivatization with cyanogen chloride and barbituric acid. This method has been used to assess the reliability of the direct barbituric acid assay to determine smoking status. It is concluded that the direct barbituric acid assay is a very reliable indicator of smoking status, provided that urine blank samples are prepared to correct for background absorbance. If the direct barbituric acid assay is in disagreement with self-reported smoking status, this HPLC procedure is a useful method to resolve the discrepancy.     

Separation and determination of liver uric acid and allantoin

We previously described the only satisfactory procedure yet achieved for separating uric acid and allantoin from rat liver. The procedure was based on trichloroacetic acid (TCA) extraction, acid hydrolysis, treatment with Hg-acetate, and cation- and anion-exchange chromatography. After separation, allantoin was quantified by a colorimetric method, and uric acid enzymatically using uricase. Since this procedure is too time-consuming, we propose an improved version which avoids the need for anion-exchange chromatography and the complex assay of catabolic compounds. The new method consists of a very fast and simple HPLC separation and direct determination of uric acid and allantoin at 220 nm. The method can be used for fresh tissue or after treatment of the tissue with labeled precursor.     

Nanoprecipitation versus emulsion-based techniques for the encapsulation of proteins into biodegradable nanoparticles and process-related stability issues

The goal of this study was to investigate the entrapment of 3 different model proteins (tetanus toxoid, lysozyme, and insulin) into poly(D,L-lactic acid) and poly(D,L-lactic-co-glycolic acid) nanoparticles and to address process-related stability issues. For that purpose, a modified nanoprecipitation method as well as 2 emulsion-based encapsulation techniques (ie, a solid-in oil-in water (s/o/w) and a double emulsion (w₁/o/w₂) method) were used. The main modification of nanoprecipitation involved the use of a wide range of miscible organic solvents such as dimethylsulfoxide and ethanol instead of the common acetone and water. The results obtained showed that tetanus toxoid and lysozyme were efficiently incorporated by the double emulsion procedure when ethyl acetate was used as solvent (>80% entrapment efficiency), whereas it was necessary to use methylene chloride to achieve high insulin entrapment efficiencies. The use of the s/o/w method or the formation of a more hydrophobic protein-surfactant ion pair did not improve protein loading. The nanoprecipitation method led to a homogenous population of small nanoparticles (with size ranging from ≈130 to 560 nm) and in some cases also improved experimental drug loadings, especially for lysozyme (entrapment efficiency >90%). With respect to drug content determination, a simple and quick matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method provided results very close to those obtained by reverse phase-high-performance liquid chromatography. With respect to protein stability, the duration and intensity of sonication were not a concern for tetanus toxoid, which retained more than 95% of its antigenicity after treatment for 1 minute. Only a high methylene chloride:water ratio was shown to slightly decrease toxoid antigenicity. Finally, no more than 3.3% of A21 desamido insulin and only traces of covalent insulin dimer were detected in nanoparticles. In conclusion, both the double emulsion and nanoprecipitation methods allowed efficient protein encapsulation. MALDI-TOF MS allowed accurate drug content determination. The manufacturing processes evaluated did not damage the primary structure of insulin. Published: December 1, 2005     

On the fidelity of deoxyribonucleic acid synthesis directed by chromatin-associated deoxyribonucleic acid polymerase beta

Accuracy of poly[d(A-T)] synthesis catalyzed by chromatin-bound deoxyribonucleic acid (DNA) polymerase beta was measured with several types. A new procedure was developed for the isolation of copied poly[d(A-T)] from chromatin DNA. This method involved in vitro copying of poly[d(A-T)] by native chromatin and subsequent selective fragmentation of chromatin by restriction nucleases, proteinase K, and heat denaturation. The fragmented natural DNA is then separated from the high molecular weight poly[d(A-T)] by gel filtration. The efficacy of DNA removal by this procedure was validated by cesium chloride gradient and nearest-neighbor analysis of the product of the reaction and by measurement of the fidelity of poly[d(A-T)] synthesis by Escherichia coli DNA Pol I contaminated with increasing amounts of DNA. Also, DNA polymerases dissociated from chromatin retain the same accuracy as that of native chromatin. Synthesis of poly[d(A-T)] by chromatin is catalyzed mainly by DNA polymerase-beta. By use of the described technique, we find that the fidelity of this reaction is exceptionally low; approximately one dGTP was incorporated for every thousand complementary nucleotides polymerized.