An antibiotic-free medium for the xenic cultivation of Entamoeba gingivalis.

Diamond's TYI-S-33 (Trypticase-Yeast Extract-Iron-Serum) medium was used as the basis for a new antibiotic-free medium for xenic growth of Entamoeba gingivalis. Nutritional requirements of the oral protozoan were determined in an effort to optimize growth. TYI-S-33 medium did not support E. gingivalis growth prior to modification. The changes included: (a) deletion of L-cysteine.HCl and thioctic acid, (b) substitution of glucose for dextran I (mol. wt 185,000) or rice starch, (c) reduction of concentrations of tryptone (2.5 g l-1), yeast extract (1.25 g l-1) and dextran I (1 g l-1), (d) increased concentration of ferric ammonium citrate (0.2 g l-1), and (e) addition of gastric mucin (2.4 g l-1). Dextran I was chosen as the major carbon source; its use in the medium limited growth of accompanying bacteria. This new antibiotic-free medium significantly increased E. gingivalis growth (16-20 E. gingivalis trophozoites observed per field) as compared to growth in Diamond's TYSGM-9 (Trypticase-Yeast Extract-Serum-Gastric Mucin) medium (six to 10 E. gingivalis trophozoites observed per field).     

Mechanisms of successive modes of erythrocyte stability and instability in the presence of various polymers

Upon examination in real time of the adhesion of human erythrocytes by observing cells suspended by ultrasonic radiation force in solutions of dextran, polylysine, and polyethylene glycol, it was reported earlier that concave-ended cell pairs and rouleaux are seen in low (0.5-2.0% w/v) concentrations of Dextran T500. At concentrations of 5-7%, dextran spherical cell doublets and convex-ended cell agglutinates are formed. When adhesion occurs in polylysine (MW 14,000) or in polyethylene glycol (MW 8,000) only spherical cell doublets or convex-ended cell clumps occur. The final cell movement completing the formation of these adhesion products takes place over time scales of the order of 1s. In this work, quantitative consideration is given to the extent to which repulsion between adhesion-inducing macromolecules associated with the glycocalyx and those free in solution can influence adhesion through a phase separation effect. It is shown for cells in dextran and in polylysine that the forces associated with this repulsion are of the same order of magnitude as the electrostatic interactions between cells.     

Structural Characteristics of Dextrans Synthesized by Dextransucrases from Leuconostoc mesenteroides NRRL B-1299

Four major dextransucrase (EC 2.4.1.5) preparations from Leuconostoc mesenteroides were studied in relation to their reaction products. The extracellular enzyme II, a highly aggregated form of enzyme I, synthesized the largest amount of dextran per 1 unit of enzyme. Moreover, this dextran emerged at the void volume by Sepharose 6B chromatography. Dextran produced by the enzyme I was composed almost exclusively of water-soluble form having a molecular weight (MW) smaller than that of the product with enzyme II. Although soluble dextran produced by the intracellular enzyme (enzyme III or IV) had a low MW, ratio of insoluble dextran to total dextran was higher than that of the products with extracellular enzyme. Dextran produced by the enzyme II contained a large amount of non-α-l,6-linkages whereas dextran produced by the enzyme I was rich in linear α-l,6-linked structure. The structural analyses of various dextrans showed that each enzyme seemed to be responsible for the synthesis of both α-1,6 and non-α-l,6-linkages. Difference in the amounts and structures of dextrans suggests that the extracellular enzymes may play a major role for the dextran synthesis in vivo.     

Mechanisms of successive modes of erythrocyte stability and instability in the presence of various polymers

Upon examination in real time of the adhesion of human erythrocytes by observing cells suspended by ultrasonic radiation force in solutions of dextran, polylysine, and polyethylene glycol, it was reported earlier that concave-ended cell pairs and rouleaux are seen in low (0.5–2.0% w/v) concentrations of Dextran T500. At concentrations of 5–7%, dextran spherical cell doublets and convex-ended cell agglutinates are formed. When adhesion occurs in polylysine (MW 14,000) or in polyethylene glycol (MW 8,000) only spherical cell doublets or convex-ended cell clumps occur. The final cell movement completing the formation of these adhesion products takes place over time scales of the order of 1s. In this work, quantitative consideration is given to the extent to which repulsion between adhesion-inducing macromolecules associated with the glycocalyx and those free in solution can influence adhesion through a phase separation effect. It is shown for cells in dextran and in polylysine that the forces associated with this repulsion are of the same order of magnitude as the electrostatic interactions between cells.     

Mechanisms of successive modes of erythrocyte stability and instability in the presence of various polymers

Upon examination in real time of the adhesion of human erythrocytes by observing cells suspended by ultrasonic radiation force in solutions of dextran, polylysine, and polyethylene glycol, it was reported earlier that concave-ended cell pairs and rouleaux are seen in low (0.5–2.0% w/v) concentrations of Dextran T500. At concentrations of 5–7%, dextran spherical cell doublets and convex-ended cell agglutinates are formed. When adhesion occurs in polylysine (MW 14,000) or in polyethylene glycol (MW 8,000) only spherical cell doublets or convex-ended cell clumps occur. The final cell movement completing the formation of these adhesion products takes place over time scales of the order of 1s. In this work, quantitative consideration is given to the extent to which repulsion between adhesion-inducing macromolecules associated with the glycocalyx and those free in solution can influence adhesion through a phase separation effect. It is shown for cells in dextran and in polylysine that the forces associated with this repulsion are of the same order of magnitude as the electrostatic interactions between cells.     

The effect of maltose on dextran yield and molecular weight distribution

Dextran synthesis has been studied since the Second World War, when it was used as blood plasma expander. This polysaccharide composed of glucose units is linked by an α-1,6-glucosidic bond. Dextransucrase is a bacterial extra cellular enzyme, which promotes the dextran synthesis from sucrose. When, besides sucrose, another substrate (acceptor) is also present in the reactor, oligosaccharides are produced and part of the glucosyl moieties from glucose is consumed to form these acceptor products, decreasing the dextran yield. Although dextran enzymatic synthesis has been extensively studied, there are few published studies regarding its molecular weight distribution. In this work, the effect of maltose on yield and dextran molecular weight synthesized using dextransucrase from Leuconostoc mesenteroides B512F, was investigated. According to the obtained results, maltose is not able to control and reduce dextran molecular weight distribution and synthesis carried out with or without maltose presented the same molecular weight distribution profile.     

Influence of medium composition on the cephalosporin C production with a highly productive strain Cephalosporium acremonium.

Cephalosporin production by a highly productive Cephalosporium acremonium strain was carried out and optimized by fed-batch operation in a 40 l stirred tank reactor using a complex medium containing 30-120 g l-1 peanut flour. The concentrations of cephalosporin C (CPC) and its precursors: penicillin N (PEN N), deacetoxy cephalosporin C (DAOC), and deacetyl cephalosporin C (DAC) were monitored with an on-line HPLC. The concentrations of amino acids valine (VAL), cysteine (CYS), alpha-amino adipic acid (alpha-AAA), the dipeptide alpha-amino-adipyl-cysteine (AC), and the tripeptide alpha-amino-adipyl-cysteinyl-valine (ACV), were determined off-line by HPLC. The RNA content and dry weight of the sediment as well as the oxygen transfer rate (OTR) and the CO2 production rate (CPR) were used to calculate the cell mass concentration (X). The influences of peanut flour (PF) and the on-line monitored and controlled medium components: glucose (GLU), phosphate, methionine (MET) as well as the dissolved oxygen (DOC) on the cell growth, the product formation, and the pathway of cephalosporin C biosynthesis were investigated and evaluated. When the glucose fed-batch cycle was optimized and oxygen transfer limitation was avoided (DOC greater than 20% of the saturation value), high process performance (103.5 g l-1 X, 11.84 g l-1 CPC, a maximum CPC productivity of 118 mg l-1 h-1, and the whole concentration of the beta-lactam antibiotics CPC, DAC, DAOC, PEN N 17.34 g l-1) was achieved by using 100 g l-1 PF in the medium with the optimum concentration of phosphate (260-270 mg l-1) and a low glucose concentration (less than 0.5 g l-1). The cultivations with different medium concentrations demonstrated that the product formation was directly proportional to the cell mass concentration. On the average, the cell mass-based yield coefficient of CPC: YCPC/X amounted to 0.115 g CPC per g cell mass.     

Analysis using a linear viscoelastic model of the in vitro osmotic kinetics of polydisperse synthetic colloids

This study clarifies the contribution to overall osmotic kinetics of colloid osmotic pressure (Pi) and the interaction of synthetic colloids with the membrane. Solutions (6%) of dextran with weight average molecular weight (MW(w)) 68 800 (DEX 70), dextran with MW(w) 40 000 (DEX 40), hydroxyethyl starch with MW(w) 70 000 (HES 70), gelatin with MW(w) 60 000 and albumin were tested. An osmotic flow cell fitted with membranes of molecular weight cutoff size 30 000 or 50 000 was used to measure time-dependent changes in Pi for each of these solutions. A linear viscoelastic model was fitted to the curve describing changes to Pi as a function of time. Values of total effective Pi for DEX 40 and DEX 70 were larger than those for HES 70, gelatin, and albumin. As an index of solute-solvent exchange rate at the membrane surface, these values were in the order DEX 40 > DEX 70, HES 70 > gelatin, albumin. The findings suggest that DEX 40 may be preferable for the temporary restoration of plasma volume because of a heightened initial osmotic force. In contrast, the osmotic force exerted by gelatin is slower to increase but is likely to be longer lasting in vivo as a result of the inhibition of gelatin from penetrating the capillary membrane due to its interaction with negatively charged groups in the endothelial glycocalyx.     

Electrically induced fusion of mammalian cells in the presence of polyethylene glycol

Chinese Hamster Ovary (CHO) cells were fused by subjecting cell suspensions to an exponentially decaying electric pulse in the presence of polyethylene glycol (PEG), Dextran or Ficoll. PEG (MW 1,000, 3,350, 8,000, 10,000 and 18,500), Dextran (MW 71,200) and Ficoll (MW 400,000) were added to the pulsing medium. A single exponential electric pulse with peak field strength of 4 kV/cm, and a half-time of 0.72 msec was used. The combination of two techniques, PEG-induced fusion and electrofusion, resulted in highly efficient fusion of CHO cells. Fusion yields (FY) at different concentrations of these polymers were measured using phase-contrast microscopy. FY was highly dependent on the concentration of PEG in media, while the presence of Dextran and Ficoll had no influence on fusion yield. PEG with MW 8,000 was found to be the most effective in causing cell aggregation, and to give the highest FY (40%). An optimal concentration for fusion was found for PEG of each molecular weight. Diluting cells suspended in higher concentrations of PEG to these optimal concentrations after the pulse application regained the optimal FY. It was concluded that PEG-induced prepulse aggregation and moderate cell swelling immediately after the pulse were important factors in achieving high fusion yields.This work is supported by a grant GM-30969 from the National Institutes of Health. Traveling fellowship to N.G.S. was supported from Foundation Cyrill and Methodius and grant N-189 from MCES of Bulgaria.     

Production & characterization of a unique dextran from an indigenous Leuconostoc mesenteroides CMG713

On the basis of high enzyme activity a newly isolated strain of L. mesenteroides CMG713 was selected for dextran production. For maximum yield of dextran, effects of various parameters such as pH, temperature, sucrose concentration and incubation period were studied. L. mesenteroides CMG713 produced maximum dextran after 20 hours of incubation at 30 masculineC with 15% sucrose at pH 7.0. The molecular mass distribution of dextran produced by this strain showed that its molecular mass was about 2.0 million Da. Dextran analysis by (13)C-NMR spectrometry showed no signals corresponding to any other linkages except alpha-(1-->6) glycosidic linkage in the main chain, which has not been reported before. Physico-chemical properties of this unique dextran were also studied. These optimised conditions could be used for the commercial production of this unique high molecular weight dextran, which have significant industrial perspectives.     

Production of cycloisomaltooligosaccharides from dextran using enzyme immobilized in multilayers onto porous membranes

Anion-exchange porous hollow-fiber membranes with a thickness of about 1.2 mm and a pore size of about 0.30 microm were used as a supporting matrix to immobilize cycloisomaltooligosaccharide glucanotransferase (CITase). CITase was immobilized to the membrane via anion-exchange adsorption and by subsequent enzymatic cross-linking with transglutaminase, the amount of which ranged from 3 to 110 mg per gram of the membrane. The degree of enzyme multilayer binding was equivalent to 0.3-9.8. Dextran, as the substrate, was converted into seven- to nine-glucose-membered cycloisomaltooligosaccharides (CI-7, -8, and -9) at a maximum yield of 28% in weight at a space velocity of 10 per hour during the permeation of 2.0% (w/w) dextran solution across the CITase-immobilized porous hollow-fiber membrane. The yield of CIs increased with increasing degree of CITase multilayering.     

Decrease of saturation density of cells of hamster cell lines after treatment with dextran sulfate

Dextran sulfates of various molecular weights were added to cultures of 3 transformed cell lines of hamster, 3T6 cells and embryonic fibroblastic cells. Dextran sulfate of high molecular weight reduced the saturation densities of all the cell lines of hamster and 3T6 cells, but those of low molecular weight did not. The mitotic rate of the treated cells decreased at stationary cell density. Dextran sulfate had no effect on the growth of normal fibroblastic cells derived from mouse and hamster embryos. Viability of treated cells was indicated by the following results. Cells of cultures seeded at different cell densities grew at almost the same rate in the presence of dextran sulfate. Treated cells remaining in the monolayer stage began to grow after removal of dextran sulfate. The colony formation rate of treated cells was the same as that of untreated cells. With the exception of one cell line, the morphology of cells treated with dextran sulfate of high molecular weight was more flattened and there was less overlapping than in untreated cells. Treated cells were less agglutinable to concanavalin A than were untreated cells. These results suggest that dextran sulfate affects the cell surface, resulting in the decrease of saturation density of cell lines.     

羰基还原酶产生菌Candida ontarioensis制备(R)-2-氯-1-(3-氯苯基)乙醇

从实验室保藏的菌株中筛选获得Candida sp.PT2A,并通过18S rRNA鉴定为安大略假单胞菌Candida on-tarioensis。对C.ontarioensis不对称还原合成(R)-2-氯-1-(3-氯苯基)乙醇的发酵产酶条件和转化条件进行优化,确定了最适的发酵产酶条件和转化条件:温度30℃,初始pH 6.5,摇床转速180 r/min,菌体质量浓度200 g/L。采用2-氯-1-(3-氯苯基)乙酮质量浓度为10 g/L时,还原反应72 h,(R)-2-氯-1-(3-氯苯基)乙醇的e.e.值为99.9%,产率为99%;底物质量浓度提高至30 g/L时,产率下降为84.3%。采用十六烷基三甲基溴化铵(CTAB)对C.ontarioensis细胞进行通透性处理(CTAB g/L,4℃下处理20 min),在30 g/L底物下反应24 h,产物的e.e.和产率分别达到99.9%和97.5%。     

Free energy potential for aggregation of erythrocytes and phosphatidylcholine/phosphatidylserine vesicles in Dextran (36,500 MW) solutions and in plasma.

The free energy potential (affinity) for aggregation of human red blood cells and lipid vesicles in Dextran solutions and blood plasma has been quantitated by measuring to what extent a vesicle is encapsulated by the red cell surface. The free energy reduction per unit area of contact formation (affinity) was computed from the observation of the fractional extent of encapsulation at equilibrium with the use of a relation based on the elastic compliance of the red cell membrane as it is deformed to adhere to the vesicle surface. Micromanipulation methods were used to select and transfer single lipid vesicles (2-3 X 10(-4) cm diameter) from a chamber that contained the vesicle suspension to a separate chamber on the microscope stage that contained red cells in an EDTA buffer with Dextran or whole plasma. The vesicle and a red cell were maneuvered into close proximity and contact allowed to take place without forcing the cells together. To evaluate the effects of surface charge density and steric interactions on aggregation, vesicles were made from mixtures of egg phosphatidylcholine (PC) and bovine phosphatidylserine (PS) over a range of mole ratios (PC/PS)from (1:0) to (1:1); the vesicles were formed by rehydration in buffer. The Dextran solutions were made with a sharp-cut fraction of 36,500 MW in a concentration range of 0-10% by weight in grams (wt/wt).(ABSTRACT TRUNCATED AT 250 WORDS)     

Blockage of undesirable endocytosis of recombinant human growth/differentiation factor-5 in Chinese hamster ovary cell cultures requires heparin analogs with specific chain lengths

Cell surface heparan sulfate proteoglycan (HSPG)-mediated endocytosis lowers the yield of recombinant human bone morphogenetic proteins (rhBMPs), such as rhBMP-2 and rhBMP-4, from Chinese hamster ovary (CHO) cell cultures. Exogenous recombinant human growth/differentiation factor-5 (rhGDF-5), a member of the BMP family, bound to cell surface HSPGs and was actively internalized into CHO cells. Knockdown of heparan sulfate (HS) synthesis enzymes in CHO cells revealed that the chain length and N-sulfation of HS affected the binding of rhGDF-5 to HSPGs and subsequent rhGDF-5 internalization. To increase product yield by minimizing rhGDF-5 internalization in recombinant CHO (rCHO) cell cultures, heparin, and dextran sulfate (DS) of various polysaccharide chain lengths, which are structural analogs of HS, were examined for blockage of rhGDF-5 internalization. Heparin fragments of four monosaccharides (MW of 1.2 kDa) and DS (MW of 15 kDa) did not inhibit rhGDF-5 internalization whereas unfractionated heparin and DS of 200 kDa could significantly inhibit it. Compared to the control cultures, supplementation with unfractionated heparin or DS of 200 kDa at 1 g L^-1 resulted in more than a 10-fold increase in the maximum rhGDF-5 concentration. Taken together, the supplementation of structural HS analogs improved rhGDF-5 production in rCHO cell cultures by inhibiting rhGDF-5 internalization.     

Molecular weight characterization of high molecular weight dextran with multiangle light scattering in on‐line and off‐line mode

This work reports the molecular weight (MW) analysis of high MW dextran using multiangle light scattering (MALS) in both chromatography and automated batch measurement mode. The results show that the chromatographic columns alter the high MW native dextran and cause underestimation of the MW as a consequence. Alternatively, a batch MALS measurement (without columns) provides more accurate MW values. The batch MALS measurement was automated with the incorporation of an automatic sample dilution and injection device. This automation reduces the sample preparation time and minimizes concentration errors introduced by manual sample dilution. To the best of our knowledge, this is the first study using an automated batch MALS in the analysis of high MW dextran. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 387–392, 2015.     

Mechanism of chain initiation by dextransucrase: Absence of terminal ‘sucrose’ linkage in newly synthesized dextran chains

Dextran was synthesized using dextransucrase from Streptococus sanguis 10558 and (F)-[¹⁴C]sucrose as substrate to test the possibility that sucrose may be the initial acceptor for glucose. If sucrose is the initial acceptor, then dextran chains should have [¹⁴C] fructose in a terminal ‘sucrose’ linkage which can be cleaved under mild conditions. Although incorporation of [¹⁴C]fructose into dextran was observed, the label was not released by mild hydrolysis, indicating that sucrose is not the initiator for dextran synthesis. Incorporation of [¹⁴C]fructose into dextran might represent its ability to act as an acceptor, as suggested by the isolation of leucrose as a by-product in the reaction.     

Dextran Nanoparticle Synthesis and Properties

Dextran is widely exploited in medical products and as a component of drug-delivering nanoparticles (NPs). Here, we tested whether dextran can serve as the main substrate of NPs and form a stable backbone. We tested dextrans with several molecular masses under several synthesis conditions to optimize NP stability. The analysis of the obtained nanoparticles showed that dextran NPs that were synthesized from 70 kDa dextran with a 5% degree of oxidation of the polysaccharide chain and 50% substitution with dodecylamine formed a NP backbone composed of modified dextran subunits, the mean diameter of which in an aqueous environment was around 100 nm. Dextran NPs could be stored in a dry state and reassembled in water. Moreover, we found that different chemical moieties (e.g., drugs such as doxorubicin) can be attached to the dextran NPs via a pH-dependent bond that allows release of the drug with lowering pH. We conclude that dextran NPs are a promising nano drug carrier.     

Influence of dissolved oxygen concentration on the biosynthesis of cephalosporin C.

Cephalosporin C was produced by a highly productive strain of Cephalosporium acremonium under industrial production conditions by fed-batch cultivation in a 40-l stirred-tank reactor using a complex medium containing 50 g l-1 peanut flour. The influence of dissolved oxygen concentration (pO2, DOC), which was maintained at different constant levels between 5 and 40% of its saturation value, during the production phase by means of a parameter-adaptive pO2-controller, on the cephalosporin C biosynthesis, was investigated. The concentrations of cephalosporin C (CPC) and its precursors penicillin N (PEN N), deacetoxycephalosporin C (DAOC), and deacetylcephalosporin C (DAC) were monitored by on-line HPLC. The concentrations of amino acids, valine (VAL), cysteine (CYS), alpha-amino-adipic acid (alpha-AAA), the dipeptide alpha-amino-adipyl-cysteine (AC), and the tripeptide alpha-amino-adipyl-cysteinyl-valine (ACV) were determined by off-line HPLC. By reducing the pO2 in the production phase from 40 to 5% of its saturation value, the CPC concentration diminished from 7.2 to 1.1 g l-1 and the PEN N concentration increased from 2.57 to 7.65 g l-1. The DAC concentration also dropped from 3.13 to 0.42 g l-1; however, the DAOC concentration was less influenced. The concentrations of AC and ACV were also less affected. The small DOC did not lead to an accumulation of the intermediate AC and ACV during the production phase. With increasing DOC in the range of 5-20%, the maximal specific production rate, the cell mass concentration-based and the substrate-based yield coefficients for CPC increased almost linearly, and fell back for PEN N.(ABSTRACT TRUNCATED AT 250 WORDS)     

Semi-quantitative assessments of dextran toxicity on corneal endothelium: conceptual design of a predictive algorithm

Dextran is added to corneal culture medium for at least 8 h prior to transplantation to ensure that the cornea is osmotically dehydrated. It is presumed that dextran has a certain toxic effect on corneal endothelium but the degree and the kinetics of this effect have not been quantified so far. We consider that such data regarding the toxicity of dextran on the corneal endothelium could have an impact on scheduling and logistics of corneal preparation in eye banking. In retrospective statistic analyses, we compared the progress of corneal endothelium (endothelium cell loss per day) of 1334 organ-cultured corneal explants in media with and without dextran. Also, the influence of donor-age, sex and cause of death on the observed dextran-mediated effect on endothelial cell counts was studied. Corneas cultured in dextran-free medium showed a mean endothelium cell count decrease of 0.7% per day. Dextran supplementation led to a mean endothelium cell loss of 2.01% per day; this reflects an increase by the factor of 2.9. The toxic impact of dextran was found to be time dependent; while the prevailing part of the effect was observed within the first 24 h after dextran-addition. Donor age, sex and cause of death did not seem to have an influence on the dextran-mediated toxicity. Based on these findings, we could design an algorithm which approximately describes the kinetics of dextran-toxicity. We reproduced the previously reported toxic effect of dextran on the corneal endothelium in vitro. Additionally, this is the first work that provides an algorithmic instrument for the semi-quantitative calculation of the putative endothelium cell count decrease in dextran containing medium for a given incubation time and could thus influence the time management and planning of corneal transplantations.