Oxidation of Se-Carboxymethyl-selenocysteine by L-aminoacid oxidase and by D-aspartate oxidase

Summary Se-Carboxymethyl-DL-selenocysteine (CMSeC) has been prepared in a pure crystalline form from selenocysteine and monochloroacetic acid. It has been shown that CMSeC is a substrate for the L-aminoacid oxidase from snake venom and for the D-aspartate oxidase from beef kidney. Oxygen consumption and ammonia production indicate that only the L or the D form of CMSeC are acted upon respectively by one or the other of the above enzymes. No noticeable differences were shown in the oxidation rate of CMSeC and S-carboxymethylcysteine, an indication that the substitution of a selenium for a sulfur atom in the molecule does not greatly affect the substrate specificity of the two enzymes. Data have been obtained suggesting that the product of the oxidative deamination of CMSeC is Se-carboxymethyl-selenopyruvic acid.     

一株纤维素分解菌的分离与筛选

以新华滤纸为唯一碳源,从垃圾堆肥中筛选能够分解纤维素的菌株共39株,采用刚果红鉴别培养基进行识别,获取透明圈较大的菌株10株,在此基础上,进行液体培养,测定酶活,得到1株酶活较高的曲霉B－6（Aspergillus sp)。将B－6与绿色木霉（Trichoderma sp)AS3.3711进行了参比试验,比对筛选工作进行评定,经过固体,液体发酵对比试验,发现B－6与AS3．371有相近的产酶性能,B－6在固,液发酵中酶活分别达到39．2IU,14．9IU,而S3．3711则分别为16．6IU与15．7IU,且B－6较AS3．3711有更强的液化CMC的能力,B－6在24h内即能使3％CMC完全液化,而S3．3711则需96h。     

Fibroblast-dependent growth of mouse mast cells in vitro: duplication of mast cell depletion in mutant mice of W/Wv genotype

In spite of the apparent depletion of mast cells in tissues of mutant mice of W/Wv genotype, cells with many features of mast cells do develop when bone marrow cells of W/Wv mice are cultured in the presence of pokeweed mitogen-stimulated spleen cell-conditioned medium (PWM-SCM). In order to resolve this discrepancy and facilitate the analysis of the W mutation, we attempted to establish an in vitro system in which the in vivo defect of W/Wv mice can be reproduced. Cultured mast cells (CMC) were developed from bone marrow cells of either W/Wv or congenic +/+ mice, and then co-cultured with NIH/3T3 mouse fibroblasts in media supplemented only with fetal calf serum (i.e., in the absence of PWM-SCM). Under this condition, CMC from +/+ mice continued to divide and were maintained for more than 4 weeks. The supportive effect of NIH/3T3 cells required close-range interactions with CMC and was not due to synthesis of the known mast cell growth factors, interleukins 3 and 4. By contrast, CMC from W/Wv mice were not maintained, and the number of mast cells remaining after 4 weeks of co-culture was only 1% of the normal +/+ counterparts. Thus, the humoral factor-independent and cell contact-dependent system presented here revealed the intrinsic defects in growth and differentiation of CMC derived from W/Wv mice and might be useful for biochemical and molecular analysis of the gene product(s) encoded at the W locus.     

The synthesis of S-(3-aminopropyl)thiosulfuric acid

In order to investigate the metabolism in vivo of homocystamine we needed the corresponding -SSO3H derivative and we attempted to prepare it. In this paper details are reported for the synthesis of S-(3-Aminopropyl)-thiosulfuric acid from 3-Bromopropylamine or thiosulfate. Same analytical date and chromatographic properties one also reported, which allow its identification.     

Biotech outsourcing strategies cmc – Biologics stream

Now in its third year, the Biotech Outsourcing Strategies (BOS) meeting organized by Bio2Business took place at the Søhuset Conference Centre in Hørsholm, Copenhagen. The focus of this year’s event was the demanding and challenging area of chemistry, manufacturing and controls (CMC), and the meeting provided ample opportunity for lively discussion of the key issues surrounding this area. New for the 2010 conference, a biologics-focused lecture stream ran in parallel to the established small molecule stream. Both streams boasted a distinguished panel of keynote speakers who discussed all aspects of CMC from early stage scale-up through late stage clinical development. In addition to the keynote speakers, selected contract research organizations (CROs) gave short presentations on the solutions that they could provide to some of the challenges facing CMC. The meeting attracted more than 150 delegates from leading drug development companies and CRO service providers, and greatly facilitated the forging of new working relationships through pre-arranged one-to-one meetings. Moreover, exhibitions from event sponsors and considerable scheduled networking time over lunch and evening reception further enhanced the highly productive and interactive nature of the meeting.     

Enzyme biotechnology development for treating polymers in hydraulic fracturing operations

Carboxymethyl cellulose (CMC) is a polymer used in many different industrial sectors. In the oil and gas industry, CMC is often used during hydraulic fracturing (fracking) operations as a thickening agent for effective proppant delivery. Accumulations of CMC at fracture faces (known as filter cakes) can impede oil and gas recovery. Although chemical oxidizers are added to disrupt these accumulations, there is industrial interest in developing alternative, enzyme-based treatments. Little is known about CMC biodegradation under fracking conditions. Here, we enriched a methanogenic CMC-degrading culture and demonstrated its ability to enzymatically utilize CMC under the conditions that typify oil fields. Using the extracellular enzyme fraction from the culture, significant CMC viscosity reduction was observed between 50 and 80˚C, at salinities up to 20% (w/v) and at pH 5–8 compared to controls. Similar levels of viscosity reduction by extracellular enzymes were observed under oxic and anoxic conditions. This proof-of-concept study demonstrates that enzyme biotechnology holds great promise as a viable approach to treating CMC filter cakes under oilfield conditions.     

一种基于胶体微晶纤维素的改良病毒蚀斑测定法的建立与评价北大核心CSCD

胶体微晶纤维素(avicel)是一种由微晶纤维素(microcrystalline cellulose, MCC)和羧甲基纤维素(carboxymethyl cellulose,CMC)制成的混合物,可用于病毒蚀斑测定。常用的avicel由FMC公司生产,其MCC和CMC比例相对固定,无法很好地适应所有类型病毒的蚀斑测定实验。本研究通过对比不同的MCC和CMC配制比例对avicel在病毒蚀斑测定作用的影响,建立了一种操作简便、实用性好和稳定性好的改良avicel病毒蚀斑测定法。为了配制不同浓度MCC和CMC的混合物,本研究制备出12种2×avicel覆盖层,测定其总体黏度及底层黏度,评估其与传统覆盖层相比,使用时的操作难易程度。进一步将12种2×avicel覆盖层制备成avicel-DMEM营养覆盖层,测定96孔板中猪流行性腹泻病毒滴度,比较12种avicel覆盖层及传统覆盖层蚀斑大小、清晰度、稳定性及滴度准确性等的差异,筛选出最佳测定方法。结果显示,12种2×avicel覆盖层中,除4.8%MCC+1.4%CMC和4.8%MCC+1.0%CMC外,其余2×avicel覆盖层在实际使用中均比2×CMC覆盖层更容易吸取和配制营养覆盖层。最后,利用avicel病毒蚀斑测定法测定96孔板中猪流行性腹泻病毒滴度,结果显示CMC浓度越高蚀斑越小,其中终浓度为0.6%MCC+0.7%CMC的avicel覆盖层测定蚀斑染色最清晰,准确度与传统覆盖层相似,但操作较传统覆盖层更简便。综上所述,本研究建立了一种操作简便、实用性好和稳定性好的改良avicel病毒蚀斑测定法,为病毒的病原学、抗病毒药物及疫苗等相关研究的展开提供了良好的实验基础。     

Studies on the reducing systems for plant and animal thioredoxin-independent methionine sulfoxide reductases B

Two distinct stereospecific methionine sulfoxide reductases (Msr), MsrA and MsrB reduce the oxidized methionine (Met), methionine sulfoxide [Met(O)], back to Met. In this report, we examined the reducing systems required for the activities of two chloroplastic MsrB enzymes (NtMsrB1 and NtMsrB2) from tobacco (Nicotiana tabacum). We found that NtMrsB1, but not NtMsrB2, could use dithiothreitol as an efficient hydrogen donor. In contrast Escherichia coli thioredoxin (Trx) could serve as a reducing agent for NtMsrB2, but not for NtMsrB1. Similar to previously reported human Trx-independent hMsrB2 and hMsrB3, NtMsrB1 could also use bovine liver thionein and selenocysteamine as reducing agents. Furthermore, the unique plant Trx-like protein CDSP32 was shown to reduce NtMsrB1, hMsrB2 and hMsrB3. All these tested Trx-independent MsrB enzymes lack an additional cysteine (resolving cysteine) that is capable of forming a disulfide bond on the enzyme during the catalytic reaction. Our results indicate that plant and animal MsrB enzymes lacking a resolving cysteine likely share a similar reaction mechanism.     

Carboxymethyl chitosan as a matrix material for platinum, gold, and silver nanoparticles

Carboxymethyl chitosan (CMC) was evaluated for its use in the synthesis and stabilization of catalytic nanoparticles for the first time. Many studies have reported on the ability of chitosan to bind with metal ions and support metal nanoparticles. CMC has a higher reported chelation capacity than chitosan, which has potential implications for improved catalyst formation and immobilization. Platinum, gold, and silver nanoparticles were synthesized in both chitosan and CMC. Particle size, morphology, and aggregation were examined using transmission electron microscopy (TEM). Complexation of nanoparticles was studied through Fourier transform infrared spectroscopy (FTIR). Similar nanoparticle size distributions were observed in the two polymers; however, CMC was observed to have higher rates of aggregation. This indicates that the carboxymethyl groups did not change nanoparticle formation; however, poor cross-linking and a limited anchoring ability of CMC led to the inability to immobilize the catalyst materials effectively.     

In vitro induction of polyclonal killer T cells with 2-mercaptoethanol and the essential role of macrophages in this process.

Killer T cells against allogeneic and syngeneic tumor cells were generated in vitro by the addition of 2-mercaptoethanol (2-ME) to the murine spleen cell culture in the absence of any antigenic stimulation. The maximum activity of T cell-mediated cytotoxicity (CMC) induced with 2-ME was observed on day 4 of culture and the induction of CMC was completely inhibited by the addition of inhibitor of DNA synthesis, hydroxyurea, or cytosin arabinoside. CMC induced with 2-ME was specifically inhibited by the addition of unlabeled target cells to the 51Cr-release assay system. These results indicated that killer T cells were generated in the presence of 2-ME as a result of nonspecific polyclonal activation of precursors into cytotoxic effector cells and that they recognized target cells with antigen-specific recognition receptors. Spleen cells deprived of adherent cells showed impaired induction of CMC with 2-ME. The addition of peritoneal exudate macrophages to splenic T cells restored this response. The result indicated that macrophages were essential for the induction of CMC with 2-ME. The possibility that the function of macrophages was mediated by soluble factor(s) released from macrophages was demonstrated by the separate culture of splenic T cells and macrophages in double-chambered, Marbrook-type vessels and by the addition of supernatants from macrophage cultures to splenic T cells. 2-ME and soluble factor(s) released from macrophages seemed to be required for the activation of precursors into killer cells.     

Acid-degradable protein delivery vehicles based on metathesis chemistry

In this communication we demonstrate that acyclic diene metathesis (ADMET) polymerization is a powerful methodology for the synthesis of acid-degradable polymers based on polyketals and polyacetals. Ten new polyketals and polyacetals were synthesized, using ADMET, and a polyacetal based on anthracene aldehyde was identified, which had the physical properties needed for microparticle formulation. The antioxidant protein catalase was encapsulated into microparticles, formulated from this polyacetal, using a double emulsion procedure, and cell culture studies demonstrated that these microparticles dramatically improved the ability of catalase to scavenge hydrogen peroxide produced by macrophages. We anticipate numerous applications of ADMET for the synthesis of acid-degradable polymers based on its excellent tolerance toward functional groups and ease of synthesis.     

5-Dodecanoylaminofluorescein as a probe for the determination of critical micelle concentration of detergents using fluorescence anisotropy

A method for determining the critical micelle concentration (CMC) of various detergents based on fluorescence polarization (anisotropy) of the lipophilic probe 5-dodecanoylaminofluorescein is presented. Nonionic, cationic, anionic, and steroid-based detergents can all be evaluated by this method and the determined CMC values of selected detergents agree well with those reported in the literature. In addition, we report the CMC of domiphen bromide, whose CMC value has not previously been described. In the case of ionic detergents, the method described is particularly sensitive at discerning changes in the CMC with increasing ionic strength of the medium and can discriminate detergent CMCs in 5 mM versus 25 mM buffering components. The described fluorescence polarization technique allows very low (submicromolar) concentrations of probe to be employed, thus minimizing the perturbation of micelle formation by 5-dodecanoylaminofluorescein insertion.     

Strategies of ultraviolet-B protection in microscopic algae

Different species of microalgae show a wide range of susceptibility to ultraviolet-B (UV-B) radiation. To identify factors responsible for the UV-B tolerance of some of the algae, we compared 8 species that are highly tolerant to UV-B to 8 species that are highly susceptible. The tolerant species contained substantial amounts of an acetolysis-resistant residue. The residue consists of sporopollenin, a biopolymer of variable chemical composition that occurs in the algal cell walls and absorbs UV-B radiation. The susceptible species contained little or no sporopollenin. We propose that sporopollenin provides protection to the tolerant species by screening the incident UV-B radiation. Previous studies showed that the mycosporine-like amino acids (MAA) also act as effective UV-B screens. Our data indicate that sporopollenin provides a constant protection while MAA are induced by radiation stress and occur with some delay. The tolerant species also differ from the susceptible species in their capacity to repair the reaction centers damaged by UV-B. The tolerant algae became vulnerable to UV-B when protein synthesis needed for repair was blocked by streptomycin. In the susceptible species, streptomycin had no effect during the UV-B stress. The repair deficiency in the susceptible species can be explained either by relatively less effective protein synthesis or by an inhibition of the protein synthesis by UV-B. In the tolerant species, the structures needed for protein synthesis are protected by UV-B screening of sporopollenin and MAA.     

Preparation and characterization of a fully active biotinylated probe of cholecystokinin

In this study we describe the synthesis and purification of biotinylated cholecystokinin-8 (Bio-CCK-8) and characterize its use as a probe for the pancreatic cholecystokinin receptor. CCK-8 (0.1 umoles) was reacted with either radiolabeled d-[8,9(-3)H]biotin succinimide ester (0.5 umoles) or N-hydroxysuccinimidyl-biotin in dimethylformamide and triethylamine, and purified by anion exchange chromatography. Concentrations of Bio-CCK-8 and CCK-8 needed for half-maximal inhibition of [125]I-CCK-8 binding to pancreatic membranes were the same (1.0 and 1.3 nM). Bio-CCK-8 retained full biological activity as determined by stimulation of pancreatic protein secretion from rats, and the biotin group bound to CCK-8 retained its high sensitivity for avidin.     

A novel approach to the measurement of surfactant parameters in arthropod digestive juices

In arthropods, the determination of two important parameters of digestive juices, i.e. the total surfactant concentration and the critical micelle concentration (CMC), is challenging due to small sample volumes and low surfactant concentrations. In this work, we report a successful implementation of potentiometric titrations using the surfactant ion-selective electrode (SISE) and the pyrene fluorescence method (PFM) for the determination of the total surfactant concentration and CMC in the digestive juice of terrestrial isopod crustaceans Porcellio scaber. Pooled digestive juice extracts of four (SISE) or two (PFM) animals were used per measurement run. In both cases, digestive juice extracts in 100 μL of deionized water were sufficient for one measurement run. The total surfactant concentration of P. scaber digestive juice was determined to be 9.2 ± 3.5 mM and the CMC was approximately 90 μM. Our work presents an important improvement towards easy CMC determination in small volume samples in comparison with the commonly used stalagmometric technique, where much larger sample volumes are usually needed. To date, the total surfactant concentration was not measured in the digestive juices of arthropods other than Homarus vulgaris, Astacus leptodactylus and Cancer pagurus, for which complex separation and analytical techniques were required. Our results obtained by SISE and PFM therefore present the first successful quantification of surfactants and their CMC in small volumes of arthropod digestive juice without prior separation or purification techniques.     

SYNTHESIS OF CELLULOSE BY ACETOBACTER XYLINUM : VIII. On the Formation and Orientation of Bacterial Cellulose Fibrils in the Presence of Acidic Polysaccharides

The transfer of the glucosyl moiety from uridine diphosphate glucose in the presence of Acetobacter xylinum cell-free extracts led to the formation of a mixture of alkali-soluble and -insoluble cellodextrins. Typical cellulose fibrils could not be detected by electron microscopy in this product. Immediately after release into the medium, cellulose formed by whole cells is in a "prefibrous" form which passes through Millipore filters of 0.45 and 0.8 µ pore diameter. Non-filtrable fibrils arise from this material probably by a process of crystallization involving no extracellular enzymes. Fibrils formed in shaken cell suspensions intertwine and form aggregates visible to the naked eye. In quiet suspensions pellicles are formed which float on the surface. Soluble Na-carboxymethylcellulose (CMC) is incorporated into cellulose fibrils formed in its presence, probably by a process of co-crystallization. Aggregation of fibrils containing CMC is delayed because of electrostatic repulsion between carboxylic groups. The aggregation time depends on the amount of CMC incorporated, its degree of substitution, the pH of the medium, and the ionic strength. The amount of CMC incorporated depends on the relative concentration CMC/cellulose and on the similarity of the CMC and the cellulose molecules i.e. in molecular weight and the number of carboxyl substitutions. Cellulose pellicles formed in the presence of CMC by unshaken cell suspensions consist of crossed, superimposed layers of parallel oriented cellulose fibrils. The same phenomenon is observed when phosphomannan, but not levan, is substituted for CMC. The biogenesis of oriented cellulose fibrils is envisaged as a process comprising the following steps: polymerization of the monomeric precursor, diffusion of the molecule to crystallization sites, crystallization, and orientation. It is proposed that charged polysaccharides play a role similar to that of CMC in affecting the orientation of cellulose fibrils in the plant cell wall.     

Liposomes as a model for the study of the mechanism of fish toxicity of sodium dodecyl sulfate in sea water.

The mechanism underlying the shark repellency of SDS was studied by comparing it with the shark nonrepelling detergent, Triton X-100. The findings can be summarized as follows: (1) The effective concentration of SDS for termination of shark tonic immobility (an immediate and fast response) was close to its critical micellar concentration in sea water (70 microM). The fish lethal concentrations (LD50) were far below the CMC value for SDS, and at CMC level for Triton X-100. (2) In sea water SDS possesses a strong affinity for lipid membranes, expressed in a lipid sea water partition coefficient (Kp) of about 3000. (3) In liposomal systems examined by assays of turbidity, fluorescence resonance energy transfer and kinetics of carboxyfluorescein (CF) release, the pattern of SDS induced changes in the phospholipid bilayer suggests: (a) absence of vesicle-vesicle fusion; (b) occurrence of vesicle size increase, and (c) nonlytic gradual release of CF above and below its CMC values. In contrast, Triton X-100 above its CMC induces membrane solubilization. (4) Assays coupling CF release from liposomes to potassium diffusion potential induced by valinomycin indicate that SDS related CF release can also be attributed to a specific mechanism such as cation pore formation and not only to membrane solubilization. The hypothesis of pore formation by SDS is discussed.     

A kinetic study of the effects of galactocerebroside 3-sulphate on human spleen glucocerebrosidase. Evidence for two activator-binding sites.

Extraction of control human spleen glucocerebrosidase with sodium cholate and butan-l-ol reversibly inactivates the enzyme in terms of its ability to hydrolyse the water-soluble substrate 4-methylumbelliferyl beta-D-glucopyranoside (MUGlc). The acidic brain lipid galactocerebroside 3-sulphate (sulphatide) reconstitutes beta-glucosidase activity in a strongly concentration-dependent manner. In this study we show that sulphatide exhibits three critical micellar concentrations (CMCs): CMC1, 3.72 microM; CMC2, 22.6 microM; CMC3, 60.7 microM. We designate the aggregates formed at these CMCs as primary, secondary and tertiary micelles respectively. From the results of kinetic studies performed at various sulphatide concentrations (0.012-248 microM), we found that sulphatide monomers (less than 3 microM) decreased the Km (for MUGlc) of control glucocerebrosidase from 11 to 4.6 mM, and lowered the Vmax. 2-fold. However, secondary and tertiary micelles were required for expression of high control glucocerebrosidase activities. Glucocerebrosidase prepared from the spleen of a patient with non-neuronopathic type 1 Gaucher's disease exhibited a very low Km (2.8 mM) even in the absence of exogenous lipid, and sulphatide monomers had no effect on the mutant enzyme's Km or Vmax. However, secondary or tertiary micelles markedly increased the Vmax. of the type 1 glucocerebrosidase to 60% of the corresponding control enzyme value. In contrast, for the glucocerebrosidase of the neuronopathic type 2 case, although sulphatide decreased the Km from 9.2 to 1.7 mM, the Vmax. never reached more than 5% that of the control enzyme, even at high concentrations of sulphatide. In addition, we found that secondary and tertiary sulphatide micelles enhanced the rate of inactivation of all three glucocerebrosidase preparations by chymotrypsin. Collectively, these results indicate the presence of two sulphatide-binding sites on glucocerebrosidase: one that enhances substrate binding, and another that enhances catalysis.     

A CMC1‐knockout reveals translation‐independent control of human mitochondrial complex IV biogenesis

Defects in mitochondrial respiratory chain complex IV (CIV) frequently cause encephalocardiomyopathies. Human CIV assembly involves 14 subunits of dual genetic origin and multiple nucleus‐encoded ancillary factors. Biogenesis of the mitochondrion‐encoded copper/heme‐containing COX1 subunit initiates the CIV assembly process. Here, we show that the intermembrane space twin CX₉C protein CMC1 forms an early CIV assembly intermediate with COX1 and two assembly factors, the cardiomyopathy proteins COA3 and COX14. A TALEN‐mediated CMC1 knockout HEK293T cell line displayed normal COX1 synthesis but decreased CIV activity owing to the instability of newly synthetized COX1. We demonstrate that CMC1 stabilizes a COX1‐COA3‐COX14 complex before the incorporation of COX4 and COX5a subunits. Additionally, we show that CMC1 acts independently of CIV assembly factors relevant to COX1 metallation (COX10, COX11, and SURF1) or late stability (MITRAC7). Furthermore, whereas human COX14 and COA3 have been proposed to affect COX1 mRNA translation, our data indicate that CMC1 regulates turnover of newly synthesized COX1 prior to and during COX1 maturation, without affecting the rate of COX1 synthesis.