Influence of thermal history on the structural and mechanical properties of agarose gels

Using a multitechnique approach, two temperature domains have been identified in agarose gelation. Below 35 degrees C, fast gelation results in strong, homogeneous and weakly turbid networks. The correlation length, evaluated from the wavelength dependence of the turbidity, is close to values of pore size reported in the literature. Above 35 degrees C, gelation is much slower and is associated with the formation of large-scale heterogeneities that can be monitored by a marked change in the wavelength dependence of turbidity and visualised by transmission electron microscopy. Curing agarose gels at temperatures above 35 degrees C, and then cooling them to 20 degrees C, produces much weaker gels than those formed directly at 20 degrees C. Dramatic reductions in the elastic modulus and failure strain and stress are found in this case as a result of demixing during cure. An interpretation, based on the kinetic competition between osmotic forces (in favor of phase separation) and elastic forces (that prevent it) is proposed.     

Transport limitation of chlorine disinfection of Pseudomonas aeruginosa entrapped in alginate beads

An artificial biofilm system consisting of Pseudomonas aeruginosa entrapped in alginate and agarose beads was used to demonstrate transport limitation of the rate of disinfection of entrapped bacteria by chlorine. Alginate gel beads with or without entrapped bacteria consumed chlorine. The specific rate of chlorine consumption increased with increasing cell loading in the gel beads and decreased with increasing bead radius. The value of an observable modulus comparing the rates of reaction and diffusion ranged from less than 0.1 to 8 depending on the bead radius and cell density. The observable modulus was largest for large (3-mm-diameter) beads with high cell loading (1.8 x 10(9) cfu/cm(3)) and smallest for small beads (0.5 mm diameter) with no cells added. A chlorine microelectrode was used to measure chlorine concentration profiles in agarose beads (3.0 mm diameter). Chlorine fully penetrated cell-free agarose beads rapidly; the concentration of chlorine at the bead center reached 50% of the bulk concentration within approximately 10 min after immersion in chlorine solution. When alginate and bacteria were incorporated into an agarose bead, pronounced chlorine concentration gradients persisted within the gel bead. Chlorine did gradually penetrate the bead, but at a greatly retarded rate; the time to reach 50% of the bulk concentration at the bead center was approximately 46 h. The overall rate of disinfection of entrapped bacteria was strongly dependent on cell density and bead radius. Small beads with low initial cell loading (0.5 mm diameter, 1.1 x 10(7) cfu/cm(3)) experienced rapid killing; viable cells could not be detected (<1.6 x 10(5) cfu/cm(3)) after 15 min of treatment in 2.5 mg/L chlorine. In contrast, the number of viable cells in larger beads with a higher initial cell density (3.0 mm diameter, 2.2 x 10(9) cfu/cm(3)) decreased only about 20% after 6 h of treatment in the same solution. Spatially nonuniform killing of bacteria within the beads was demonstrated by measuring the transient release of viable cells during dissolution of the beads. Bacteria were killed preferentially near the bead surface. Experimental results were consistent with transport limitation of the penetration of chlorine into the artificial biofilm arising from a reaction-diffusion interaction. The methods reported here provide tools for diagnosing the mechanism of biofilm resistance to reactive antimicrobial agents in such applications as the treatment of drinking and cooling waters. (c) 1996 John Wiley & Sons, Inc.     

Temperature-sensitive expression of all-Torpedo and Torpedo-rat hybrid AChR in mammalian muscle cells

When the four subunits of the Torpedo californica nicotinic acetylcholine receptor (AChR) are expressed in mammalian fibroblasts, they properly assembly into alpha 2 beta gamma delta pentamers only at temperatures lower than 37 degrees C (Claudio, T., W. N. Green, D. S. Hartman, D. Hayden, H. L. Paulson, F. J. Sigworth, S. M. Sine, and A. Swedlund. 1987. Science (Wash. DC). 238:1688-1694). Experiments here with rat L6 myoblast cell lines indicate that this temperature sensitivity is not specific to fibroblasts, but is intrinsic to Torpedo subunits. A clonal isolate of L6 cells cotransfected with the four Torpedo subunit cDNAs synthesizes the exogenous AChR subunits at 37 degrees and 26 degrees C, but expresses Torpedo AChR complexes only at the lower temperature. When Torpedo alpha alone is expressed in L6 myotubes, hybrid AChRs are formed, again only at temperatures below 37 degrees C. These hybrid AChRs can contain either two Torpedo alpha subunits or one each of rat and Torpedo alpha, proving that the two alpha subunits in an AChR pentamer need not derive from the same polysome. Further analysis of hybrid and all-Torpedo AChR established that there is no internally sequestered pool of AChR at the nonpermissive temperature, and that the AChR, once formed, is thermostable. Two lines of experimentation with alpha subunits expressed in fibroblasts indicate that alpha polypeptides exhibit different conformations at 26 degrees and 37 degrees C, favoring the hypothesis that the temperature-sensitive step occurs before assembly and reflects, at least in part, misfolding of subunits: at 37 degrees C, there is a reduction in the fraction of alpha subunits that (a) bind the AChR antagonist alpha-bungarotoxin with high affinity; and (b) bind a monoclonal antibody that recognizes correctly folded and/or assembled alpha subunit.     

Separation and recovery of DNA fragments by electrophoresis through a thermoreversible hydrogel composed of poly(ethylene oxide) and poly(propylene oxide)

We have synthesized and characterized a thermoreversible hydrogel of multiplied block copolymers, composed of poly(ethylene oxide) and poly(propylene oxide), for DNA electrophoresis. The aqueous solution of block copolymers turned into a hydrogel upon heating at temperatures above 10-11 degrees C, whereas it reverted into a solution upon cooling. Linear double-stranded DNA molecules migrated through the gel matrices at a rate that was inversely proportional to the logarithm of the DNA length. The hydrogel is most effective for separating DNA fragments in the 10- to 2000-bp range. The resolving range lay in-between the effective ranges of polyacrylamide and agarose gel electrophoreses of DNA. The gel slices containing DNA fragments were liquefied by cooling on ice, and the DNA was precipitated with ethanol. No contaminants that inhibit enzymatic reactions were found in the DNA recovered from the hydrogel. Plasmid DNA recovered from the hydrogel was recircularized with T4 DNA ligase and yielded highly efficient Escherichia coli transformation. Therefore, thermoreversible gel electrophoresis will be a useful method for DNA separation and isolation in recombinant DNA technology.     

可用于微生物群落分子生态学研究的活性污泥总DNA提取方法研究

活性污泥样品经液氮速冻、沸水浴融化、溶菌酶处理和 SDS裂解后 ,99%以上细胞裂解。所提取的 DNA经琼脂糖凝胶电泳检测和荧光法浓度测定 ,其片断大小在 2 0 kb左右 ,产量可达 1 .75 6± 0 .1 mg/g MLSS。样品 ABS2 6 0 nm/ABS2 80 nm的比值为 1 .96± 0 .2。以提取的总 DNA为模板 ,进行细菌核糖体小亚基 1 6Sr DNA基因 V3区和多组分苯酚羟化酶大亚基基因 (Lm PHs)的 PCR扩增 ,均获得成功 ,为活性污泥中微生物群落的分子生态学研究提供了一种简便、可靠的 DNA提取方法。     

人胚胎干细胞程序降温保存的实验研究

本文采用升降式程序降温仪对人胚胎干细胞进行了程序降温保存,并探讨和比较了降温速率、置核温度、保护剂和投入液氮前温度对冻存复苏后胚胎干细胞的存活率、活力及分化特性的影响。结果表明：采用Me2SO 血清 DMEM(体积比为1：3：6)的保护剂,从0℃开始,以0．5℃／min的速率对细胞悬液降温;至-10℃时对其进行置核,并于-35℃时将其快速投入液氮中保存,复温后效果最佳。冻存复温后细胞存活率可达81．8％,复苏后的胚胎干细胞形态和集落生长方式都与冻前的生长形态相同,且胚胎干细胞标志之一碱性磷酸酶(AKP)反应阳性,同时染色体组型仍正常。     

Determination of hematocrit based on diffusion of an inert molecular probe from agarose gels into whole blood

Whole blood hematocrit was determined by an approach which depends on the diffusion of an inert probe, to which red blood cells are impermeable, from a small agarose gel into a stirred, much larger blood sample. Blood cells influence the diffusion rate of the probe by, on the average, physically blocking a fraction of the gel surface. The blocking effect increases with the hematocrit. Cyanocobalamin (B-12) was found to be a suitable probe because it did not penetrate, bind to, or lyse blood cells and was not bound by plasma solutes. The loss of B-12 from gels in contact with blood was monitored by determination of the absorbance change at 540 nm of gels which had been quickly rinsed. The visible spectrum of B-12 in agarose gels was identical to the spectrum in water. Beer's Law was obeyed in 1-mm thick agarose gels over a concentration range of 0.1-0.8 mM. Based on the results from 48 blood samples covering the hematocrit range 25-69, a least-squares line was generated with a slope, -3.46 X 10(-3) delta A/hematocrit unit, a Y intercept of 0.295, and a correlation coefficient of 0.971. The precision of the technique was +/- 9.7%. The assay was insensitive to mean corpuscular volume and sample volume as long as the latter was 50-fold larger than the gel volume. The diffusion coefficient for B-12 in 1% agarose gels was found to be 1.4 +/- 0.2 X 10(-6) cm2 sec-1.     

Membrane integrity and development of immature murine cumulus-oocyte complexes following slow cooling to -60 degrees C: the effect of immediate rewarming, plunging into LN2 and two-controlled-rate-stage cooling

Cryopreservation of murine germinal vesicle (GV) stage cumulus-oocyte complexes (COCs) has been shown to result in poor development and cumulus cell damage. In an attempt to determine the stage of the cryopreservation protocol at which damage occurs, three cooling profiles were compared: slow-cooling (0.3 degrees C/min) to -60 degrees C (protocol A); slow-cooling to -60 degrees C and plunging to -196 degrees C (protocol B); or slow-cooling to -60 degrees C followed by further cooling at 10 degrees C/min to -150 degrees C, then plunging to -196 degrees C (protocol C). GV-stage COCs were collected from hormone-primed mice by repeated puncturing of ovarian follicles. COCs were exposed to 1.5 M Me(2)SO prior to cooling to -60 or -196 degrees C. Membrane integrity was assessed immediately after thawing using carboxy fluorescein and propidium iodide. A greater proportion of cumulus cells were damaged following protocol B than protocol A. Damage was less extensive following protocol C than following protocol B. For assessment of development, COCs were matured and fertilised in vitro. Morphological normality was significantly reduced following cooling to -60 or -196 degrees C compared with non-cryopreserved controls. Fertilisation of oocytes assessed as normal post-treatment was not significantly different between any of the groups. Development to blastocyst was least from oocytes exposed to protocol B, being significantly worse than for oocytes exposed to protocol A, but not significantly different to protocol C. A protocol comprising two stages of controlled-rate cooling decreased damage to the membranes of cumulus cells but did not significantly improve embryo development.     

Continuous perfusion of mammalian cells embedded in agarose gel threads

A method for perfusing cells by embedding them in fine agarose gel threads is described and characterized. The rate of diffusion of a metabolite into the gel threads is determined by 31P-NMR spectroscopy. This perfusion method is shown to enable Chinese hamster lung fibroblasts (CHLF) to remain in a metabolically active state with high levels of intracellular ATP for many hours.     

The structure and thermotropic phase behaviour of dipalmitoylphosphatidylcholine codispersed with a branched-chain phosphatidylcholine

The structure and thermotropic phase behaviour of a fully hydrated binary mixture of dipalmitoylphosphatidylcholine and a branched-chain phosphatidylcholine, 1, 2-di(4-dodecyl-palmitoyl)-sn-glycero-3-phosphocholine, were examined using differential scanning calorimetry, synchrotron X-ray diffraction and freeze-fracture electron microscopy. The branched-chain lipid forms a nonlamellar phase when dispersed alone in aqueous medium. Mixed aqueous dispersions of the two phospholipids containing less than 33 mol% of the branched-chain lipid form lamellar phases over the whole temperature range were studied (4 degrees C to 60 degrees C). When present in proportions greater than 33 mol% it induces a hexagonal phase in mixed aqueous dispersions with dipalmitoylphosphatidylcholine at temperatures above the fluid phase transition. At temperatures below 35 degrees C a hexagonal phase coexists with a gel bilayer phase. The lamellar<-->nonlamellar transition can be explained satisfactorily on the basis of the shape of the molecule expressed in terms of headgroup and chain cross-sectional areas. At temperatures below 35 degrees C macroscopic phase separation of two gel phases takes place. Freeze-fracture electron microscopy revealed that one gel phase consists of bilayers with a highly regular, periodic superstructure (macro-ripples) whereas the other phase forms flat, planar bilayers. The macro-ripple phase appears to represent a relaxation structure required to adapt to the packing constraints imposed by the incorporation of the branched-chain lipid into the dipalmitoylphosphatidylcholine host bilayer. The data suggest that structural changes that take place on cooling the mixed dispersion below the lamellar<-->nonlamellar phase transition temperature cannot be adequately described using the molecular form concept. Instead it is necessary to take into account the detailed molecular form of the guest lipid as well as its physical properties.     

Manifestations of cell damage after freezing and thawing

The nature of the primary lesions suffered by cells during freezing and thawing is unclear, although the plasma membrane is often considered the primary site for freezing injury. This study was designed to investigate the nature of damage immediately after thawing, by monitoring several functional tests of the cell and the plasma membrane. Hamster fibroblasts, human lymphocytes, and human granulocytes were subjected to a graded freeze-thaw stress in the absence of cryoprotective compound by cooling at -1 degree C/min to a temperature between -10 and -40 degrees C, and then were either warmed directly in water at 37 degrees C or cooled rapidly to -196 degrees C before rapid warming. Mitochondrial function in the cells was then assessed using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT), fluorescein diacetate (FDA), colony growth, and osmometric response in a hypertonic solution. Cells behaved as osmometers after cooling at -1 degree C/min to low temperatures at which there were no responses measured by other assays, indicating that the plasma membrane is not a primary site for injury sustained during slow cooling. These results also indicate that the FDA test does not measure membrane integrity, but reflects the permeability of the channels through which fluorescein leaves the cells. Fewer cells could respond osmotically after cooling under conditions where intracellular freezing was likely, implying that the plasma membrane is directly damaged by the conditions leading to intracellular freezing. A general model of freezing injury to nucleated mammalian cells is proposed in which disruption of the lysosomes constitutes the primary lesion in cells cooled under conditions where the cells are dehydrated at low temperatures.     

Novel thermoresponsive nonviral gene vector: P(NIPAAm-co-NDAPM)-b-PEI with adjustable gene transfection efficiency

A thermoresponsive cationic copolymer, poly( N-isopropylacrylamide- co- N-(3-(dimethylamino)propyl)methacrylamide)- b-polyethyleneimine (P(NIPAAm- co-NDAPM)- b-PEI), was designed and synthesized as a potential nonviral gene vector. The lower critical solution temperature (LCST) of P(NIPAAm- co-NDAPM)- b-PEI in water measured by UV-vis spectroscopy was 38 degrees C. P(NIPAAm- co-NDAPM)- b-PEI as the gene vector was evaluated in terms of cytotoxicity, buffer capability determined by acid-base titration, DNA binding capability characterized by agarose gel electrophoresis and particle size analysis, and in vitro gene transfection. P(NIPAAm- co-NDAPM)- b-PEI copolymer exhibited lower cytotoxicity in comparison with 25 kDa PEI. Gel retardation assay study indicated that the copolymer was able to bind DNA completely at N/P ratios higher than 30. At 27 degrees C, the mean particle sizes of P(NIPAAm- co-NDAPM)- b-PEI/DNA complexes decreased from 1200 to 570 nm corresponding to the increase in N/P ratios from 10 to 60. When the temperature changed to 37 degrees C, the mean particle sizes of complexes decreased from 850 to 450 nm correspondingly within the same N/P ratio range due to the collapse of thermoresponsive PNIPAAm segments. It was found that the transfection efficiency of P(NIPAAm- co-NDAPM)- b-PEI/DNA complexes was higher than or comparable to that of 25 kDa PEI/DNA complexes at their optimal N/P ratios. Importantly, the transfection efficiency of P(NIPAAm- co-NDAPM)- b-PEI/DNA complexes could be adjusted by altering the transfection and cell culture temperature.     

Effect of annealing time of an ice crystal on the activity of type III antifreeze protein

Antifreeze proteins (AFPs) possess a unique ability to bind to a seed ice crystal to inhibit its growth. The strength of this binding has been evaluated by thermal hysteresis (TH). In this study, we examined the dependence of TH on experimental parameters, including cooling rate, annealing time, annealing temperature and the size of the seed ice crystal for an isoform of type III AFP from notched-fin eelpout (nfeAFP8). TH of nfeAFP8 dramatically decreased when using a fast cooling rate (0.20 degrees C x min(-1)). It also decreased with increasing seed crystal size under a slow cooling rate (0.01 degrees C x min(-1)), but such dependence was not detected under the fast cooling rate. TH was enhanced 1.4- and 2.5-fold when ice crystals were annealed for 3 h at 0.05 and 0.25 degrees C below T(m), respectively. After annealing for 2 h at 0.25 degrees C below T(m), TH activity showed marked dependence on the size of ice crystals. These results suggest that annealing of an ice crystal for 2-3 h significantly increased the TH value of type III AFP. Based on a proposed adsorption-inhibition model, we assume that type III AFP undergoes additional ice binding to the convex ice front over a 2-3 h time scale, which results in the TH dependence on the annealing time.     

Cryopreservation of tissue-engineered dermal replacement in Me2SO: Toxicity study and effects of concentration and cooling rates on cell viability

Cryopreservation of tissue-engineered human dermal replacement plays an important role in skin tissue engineering and skin banking. With the inspection of electronic scanning microscope and viability evaluation by Trypan Blue staining assay and the tetrazolium salt, MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, this study investigated the toxicity of Me(2)SO to dermal fibroblasts and effects of cryoprotectant concentration and cooling rate on the viability of dermal replacement. The results demonstrated that the Me(2)SO toxicity to fibroblasts was affected by the exposure time, temperature, and concentration. Furthermore adding cryoprotectant solution at low temperature of 4 degrees C significantly reduced the toxic effect on the tissue-engineered dermal equivalent. An optimal cryopreservation protocol consisting of cooling rate at 1 degrees Cmin(-1) in 10% (V/V) Me(2)SO was derived, with the viability of studied dermal equivalent treated by this protocol being 75% of that of fresh control. The micrograph obtained by electronic scanning microscope also confirmed this result.     

An improved plaque assay for viruses infecting poikilothermic cells; use of ultralow-gelling-temperature agarose

The conventional plaque assay for viruses infecting poikilothermic cells is difficult because the cells are easily damaged at temperatures needed to keep the agarose overlay from solidifying prematurely. A modification of the assay in which these problems were solved by use of a new type of agarose (SeaPrep 15/45) that remains liquid above 15 degrees C, was developed. Use of this agarose made it easy to obtain smooth agar overlays without the risk of thermal damage to the cells.     

Single step synthesis and characterization of thermoresponsive hyaluronan hydrogels

An efficient and scale-up ready single-step synthesis for the conjugation of thermoresponsive polymers to hyaluronic acid (HA) was established. Jeffamines(?) (JFM) and poly(N-isopropylacrylamide) (PNIPAM) were grafted to HA via direct amidation mediated by 1,1'-carbonyldiimidazole activation. The temperature-induced gelation of the semi-synthetic co-polymers was characterized by rheology as a function of the temperature and by differential scanning calorimetry (DSC). A HA-JFM conjugate with sol-gel transition in a physiologically relevant temperature range was identified. The grafting of PNIPAM resulted in the drastic change of the main rheological properties of native HA, revealing the hydrophobic non-covalent nature of the interactions between the thermoresponsive brushes in the gel state. Owing to the reversibility of these interactions and the sharpness of the transition, the HA-PNIPAM conjugates are suitable candidates for the incorporation of drugs, cells or ceramic materials for different biomedical applications.     

Enzymatic liquefaction of agarose above the sol–gel transition temperature using a thermostable endo-type β-agarase,Aga16B

The main carbohydrate of red macroalgae is agarose, a heterogeneous polysaccharide composed of d-galactose and 3,6-anhydro-l-galactose. When saccharifying agarose by enzymes, the unique physical properties of agarose, namely the sol–gel transition and the near-insolubility of agarose in water, limit the accessibility of agarose to the enzymes. Due to the lower accessibility of agarose to enzymes in the gel state than to the sol state, it is important to prevent the sol–gel transition by performing the enzymatic liquefaction of agarose at a temperature higher than the sol–gel transition temperature of agarose. In this study, a thermostable endo-type β-agarase, Aga16B, originating from Saccharophagus degradans 2-40^T, was characterized and introduced in the liquefaction process. Aga16B was thermostable up to 50 °C and depolymerized agarose mainly into neoagarooligosaccharides with degrees of polymerization 4 and 6. Aga16B was applied to enzymatic liquefaction of agarose at 45 °C, which was above the sol–gel transition temperature of 1 % (w/v) agarose (∼35 °C) when cooling agarose. This is the first systematic demonstration of enzymatic liquefaction of agarose, enabled by determining the sol–gel temperature of agarose under specific conditions and by characterizing the thermostability of an endo-type β-agarase.

     

Stabilization of frozen Lactobacillus delbrueckii subsp. bulgaricus in glycerol suspensions: Freezing kinetics and storage temperature effects

The interactions between freezing kinetics and subsequent storage temperatures and their effects on the biological activity of lactic acid bacteria have not been examined in studies to date. This paper investigates the effects of three freezing protocols and two storage temperatures on the viability and acidification activity of Lactobacillus delbrueckii subsp. bulgaricus CFL1 in the presence of glycerol. Samples were examined at -196 degrees C and -20 degrees C by freeze fracture and freeze substitution electron microscopy. Differential scanning calorimetry was used to measure proportions of ice and glass transition temperatures for each freezing condition tested. Following storage at low temperatures (-196 degrees C and -80 degrees C), the viability and acidification activity of L. delbrueckii subsp. bulgaricus decreased after freezing and were strongly dependent on freezing kinetics. High cooling rates obtained by direct immersion in liquid nitrogen resulted in the minimum loss of acidification activity and viability. The amount of ice formed in the freeze-concentrated matrix was determined by the freezing protocol, but no intracellular ice was observed in cells suspended in glycerol at any cooling rate. For samples stored at -20 degrees C, the maximum loss of viability and acidification activity was observed with rapidly cooled cells. By scanning electron microscopy, these cells were not observed to contain intracellular ice, and they were observed to be plasmolyzed. It is suggested that the cell damage which occurs in rapidly cooled cells during storage at high subzero temperatures is caused by an osmotic imbalance during warming, not the formation of intracellular ice.     

PCR-amplification of GC-rich regions: 'slowdown PCR'

The polymerase chain reaction (PCR) technique has become an indispensable method in molecular research. However, PCR-amplification of GC-rich templates is often hampered by the formation of secondary structures like hairpins and higher melting temperatures. We present a novel method termed 'Slowdown PCR', which allows the successful PCR-amplification of extremely GC-rich (>83%) DNA targets. The protocol relies on the addition of 7-deaza-2'-deoxyguanosine, a dGTP analog to the PCR mixture and a novel standardized cycling protocol with varying temperatures. The latter consists of a generally lowered ramp rate of 2.5 degrees C s(-1) and a low cooling rate of 1.5 degrees C s(-1) for reaching an annealing temperature and is run for 48 cycles. We established this protocol as a versatile method not only for amplification of extremely GC-rich regions, but also for routine DNA diagnostics and pharmacogenetics for templates with different annealing temperatures. The protocol takes 5 h to complete.     

Affinity purification methods. Improved procedures for cyanogen bromide reaction on agarose.

The high solubility of cyanogen bromide inN-methyl-2-pyrrolidone has afforded an efficient means of controlling the amount of the reagent used to activate an agarose preparation. By controlling the activation step, one can determine the final concentration of the leash moiety. The upper limits of ε-aminocaproic acid incorporation seem to be about 5, 20, and 60 μequiv/ml gel, respectively, for 2, 4, and 6% agarose gels. It is important to conduct the cyanogen bromide reaction at 4–10°C, but the coupling step may be done at room temperature.