An evaluation of colloidal solutions for normothermic perfusion of rabbit hearts: An improved perfusate containing Haemaccel

Rabbit hearts were perfused at 37 °C with the aim of establishing a relatively simple and preferably synthetic perfusate which would give reliable and consistent perfusion performances for periods of several hours. Of the perfusates devised and tested, a 1.75% solution of Haemaccel most fully satisfied the above criteria and maintained myocardial function for 30.0 ± 4.0 hr.     

An Analysis of the Surface Fixed-Charge Theory of the Squid Giant Axon Membrane

The observed shift in threshold potential, after perfusion of the squid giant axon with solutions of low ionic strength, can be predicted by assuming a fixed negative charge on the inside of the membrane. The constant field equation, together with the double-layer potential due to this charge, has been used to determine the change in resting potential during perfusion with solutions of low ionic strength. Neither the modified constant field equation nor Planck's diffusion equation can successfully predict the observed shift in resting potential. It is suggested that a positive charge distribution exists about the sodium channel on the outside of the membrane. The double-layer potential due to this positive charge, together with the independence principle, has been used to predict the relationship between sodium current and membrane potential when the ionic strength and sodium activity of the external solution are decreased. These predictions have been compared with the available experimental observations.     

A comparison of microwave heating and proteolytic pretreatment antigen retrieval techniques in formalin fixed, paraffin embedded tissues

Antigen retrieval (AR) is a technique that re-exposes epitopes in formalin fixed, paraffin embedded sections and makes them detectable by immunohistochemistry. We compared the effects of two AR procedures, enzyme digestion and microwave heating, on immunostaining of vimentin and desmin in formalin fixed, paraffin embedded tissues. Our results showed that AR is necessary for vimentin and desmin immunostaining in tissues fixed in formalin for more than 48 h. With prolonged fixation times, microwave heating showed better results than enzyme digestion for AR. The same results were obtained using 1% zinc sulfate or Citra Plus solution as retrieval solutions for microwave heating. We recommend microwave heating for AR, because it is easier to use and produces better results compared to enzyme treatment.     

A comparison of microwave heating and proteolytic pretreatment antigen retrieval techniques in formalin fixed,paraffin embedded tissues

Antigen retrieval (AR) is a technique that re-exposes epitopes in formalin fixed, paraffin embedded sections and makes them detectable by immunohistochemistry. We compared the effects of two AR procedures, enzyme digestion and microwave heating, on immunostaining of vimentin and desmin in formalin fixed, paraffin embedded tissues. Our results showed that AR is necessary for vimentin and desmin immunostaining in tissues fixed in formalin for more than 48 h. With prolonged fixation times, microwave heating showed better results than enzyme digestion for AR. The same results were obtained using 1% zinc sulfate or Citra Plus solution as retrieval solutions for microwave heating. We recommend microwave heating for AR, because it is easier to use and produces better results compared to enzyme treatment.     

Transport studies with brush border membrane vesicles: choice of experimental parameters

1. We have studied different parameters, in their effects on a transport system chosen as a model: the Na+-phosphate symporter of the renal brush border membrane. 2. Ionic strength was found to be a critical factor in the retention capacity of the filter. 3. When high ionic strength solutions containing 150 mM NaCl or KCl were used, less than 8% of the membrane proteins were lost through filtration. 4. Lowering the ionic strength by replacing NaCl or KCl by 300 mM mannitol, however, caused a 52% loss of protein. 5. Addition of 15 mM NaCl to this low ionic strength solution was sufficient to restore full retention of the vesicles by the filter. 6. The presence of arsenate, a competitive inhibitor, in the stop solution did not improve the retention of phosphate by the vesicles in high ionic strength media, but caused a pronounced temperature dependent loss of the vesicle content, as a function of time of incubation in low ionic strength solutions. 7. Addition of 5 mM phosphate in the stop solution caused a 31 and 37% loss for KCl and NaCl stop solutions, respectively, while no effect was observed for the mannitol stop solution. 8. The presence of HgCl2 gave a 32% stimulation for the mannitol solution and a 35 or 22% inhibition for the KCl or NaCl solutions. 9. Addition of NaCl in the stop solution caused an overaccumulation of 75%, after 60 sec of incubation at 25 degrees C. 10. Phosphate transport by renal vesicles is thus highly affected by the composition of the stop solution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stability of Secondary and Tertiary Structures of Virus-Like Particles Representing Noroviruses: Effects of pH,Ionic Strength,and Temperature and Implications for Adhesion to Surfaces

Loss of ordered molecular structure in proteins is known to increase their adhesion to surfaces. The aim of this work was to study the stability of norovirus secondary and tertiary structures and its implications for viral adhesion to fresh foods and agrifood surfaces. The pH, ionic strength, and temperature conditions studied correspond to those prevalent in the principal vehicles of viral transmission (vomit and feces) and in the food processing and handling environment (pasteurization and refrigeration). The structures of virus-like particles representing GI.1, GII.4, and feline calicivirus (FCV) were studied using circular dichroism and intrinsic UV fluorescence. The particles were remarkably stable under most of the conditions. However, heating to 65°C caused losses of β-strand structure, notably in GI.1 and FCV, while at 75°C the α-helix content of GII.4 and FCV decreased and tertiary structures unfolded in all three cases. Combining temperature with pH or ionic strength caused variable losses of structure depending on the particle type. Regardless of pH, heating to pasteurization temperatures or higher would be required to increase GII.4 and FCV adhesion, while either low or high temperatures would favor GI.1 adhesion. Regardless of temperature, increased ionic strength would increase GII.4 adhesion but would decrease GI.1 adhesion. FCV adsorption would be greater at refrigeration, pasteurization, or high temperature combined with a low salt concentration or at a higher NaCl concentration regardless of temperature. Norovirus adhesion mediated by hydrophobic interaction may depend on hydrophobic residues normally exposed on the capsid surface at pH 3, pH 8, physiological ionic strength, and low temperature, while at pasteurization temperatures it may rely more on buried hydrophobic residues exposed upon structural rearrangement.     

Preparation of clear solutions of reconstituted acetylcholinesterase. Effect of ionic strength and lipid/protein ratio

The detergent-soluble globular dimer of acetylcholinesterase from Torpedo californica was reconstituted through dialysis into preformed egg phosphatidylcholine vesicles. The formation of the enzyme-lipid complexes depended on the ionic strength of the dialysis buffer as well as the molar lipid/protein ratio (R). The enzyme was unstable at I less than 0.05; increasing the ionic strength increased the size of the complex. A too low R value (e.g. 1000) would promote self-aggregation of the enzyme and produce heterogeneous complexes, especially at high I values. On the other hand, a too high R value (e.g. greater than 5000) favored the formation of large enzyme-lipid complexes; their solutions were too turbid for optical studies. The enzyme reconstituted at I = 0.07 and R = 4000 gave a clear solution and showed no artifacts due to light scattering. The conformation based on circular dichroism and enzymatic activity of the detergent-soluble enzyme were unchanged upon reconstitution. The reconstituted enzyme in lipid vesicles seemed to be slightly more stable against thermal denaturation than the protein in sodium cholate solution.     

Comparative Study of Antigen Retrieval Heating Methods: Microwave, Microwave and Pressure Cooker, Autoclave, and Steamer

We present a study comparing the most popular beating methods currently used for antigen retrieval (AR) immunostaining: the microwave oven, microwave with pressure cooker, autoclave, and steamer heating. A panel of 21 antibodies was tested on formalin fixed, paraffin embedded sections using these heating methods and Tris-HC1 buffer, pH 9.5, plus 5% urea as the AR solution. Three observers independently evaluated the intensity of AR immunostaining. All heating methods yielded good results for AR immunostaining. There were only minor differences among the heating methods for AR when the optimal concentration of primary antibody for normal immunostaining was used; however, background staining may occasionally be troublesome if antibodies are not retitrated and diluted further for use on tissues after AR. Significant differences were observed only after further dilution of the primary antibodies; the microwave pressure cooker, extended microwave heating (5 min × 4) and autoclave heating then showed a similar intensity of staining that was stronger than results obtained with the steamer (20 min) or regular microwave heating (5 min × 2 J. Extension of the steamer heating time, however, yielded equivalent results. This study indicates that different heating methods can yield similar intensities of AR immunostaining if the heating times are adjusted appropriately. It is noteworthy that, in general, the adjusted conditions for maximal retrieval differ from those most widely cited in the literature, or recommended by manufacturers. That several heating devices may provide similar results permits the use of different AR heating methods according to the equipment available. This study also is an early step in standardizing the AR immunostaining protocol by providing uniform conditions for “maximal retrieval” as a common end point for all laboratories.     

Physicochemical Properties and Heat-induced Gelling of Cardiac Myosin in Model System

Some physicochemical and functional properties of cardiac myosin were studied in a model system, with particular reference to its binding ability in re-structured meat. We found that myosin solubility was strongly influenced by the pH, ionic strength, and temperature of the system and by the interaction of pH and ionic strength. For instance, myosin remained completely in solution in monomeric form at ionic strengths ≤0.2 M KCl, if the pH of system was maintained at 7.0. Highionic strength was required to keep myosin in monomeric form at low pH. With low ionic strength and pH, myosin molecules tend to form aggregated filaments.

Like skeletal muscle myosin, the heat-induced gel strength of cardiac myosin was also influenced by the pH, ionic strength, and temperature of the system, and it produced a gel with maximum strength (21.× 10³dyn/cm²) at pH 5.5 and 0.1 M KCl concentration on heating to 60%C. Cardiac myosin seems to form much stronger gels than skeletal muscle myosin.     

Species-dependent differences in the effect of ionic strength on potassium transport of erythrocytes: the role of lipid composition.

The Rb+(K+) efflux of erythrocytes from six mammalian species was investigated in solutions of physiological and low ionic strength. A species dependent increase of the Rb+(K+) efflux in low ionic strength solution could be observed. The rate constant of Rb+(K+) efflux of erythrocytes in physiological ionic strength solution correlates with the content of arachidonic acid of the membrane phospholipids. The same relation was observed in solution of low ionic strength with the exception of human erythrocytes. In addition, an age-dependent correlation of the rate constant of Rb+(K+) efflux from calf erythrocytes in low ionic strength solution with the content of arachidonic acid of the membrane phospholipids was found. The Rb+(K+) efflux of human erythrocytes, which is enhanced in low ionic strength solution, decreases with the decreasing temperature. The temperature-dependent ESR order parameter of a fatty acid spin label for human and cow erythrocytes in solution of physiological and low ionic strength media suggested that the effect of low ionic strength on Rb+(K+) efflux is not solely based on a change of membrane fluidity. The results are interpreted as being due to a specific influence of membrane phospholipids on the Rb+(K+) efflux.     

Continuous enzymatic cooking and liquefaction of starch using the technique of direct resistive heating

Continuous cooker prototypes of very simple design, using electricity as a primary energy source, were developed for the process of cooking and liquefaction of starch suspensions. Previous work on equipment using microwave dielectric heating has already been reported. Results of energy consumption as low as 330 kcal/kg based on starch content were achieved. Considering these results and looking for new solutions or engineering concepts, the authors have been investigating the possibility of using electric energy at 60 Hz for direct resistive heating, in which the starch suspension is the proper "resistor."The most important results of energetic yield obtained until now, working in a continuous process of cooking-liquefaction, are not larger than 235 kcal (272 Wh)/kg based on starch content. These results were obtained using a commercial grade alpha-amylase from B. subtillis, working with temperatures ranging from 70 to 75 degrees C, and with residence times in the reactor not greater than 1.5 min. The experiments of saccharification and fermentation accomplished as a test for the efficiency of this heating technique gave good results (as with a conventional technique) and thus enabled us to proceed with the studies.     

An approach to hypothermic renal preservation

A selective review of the literature concerning hypothermic renal storage suggests that two major interrelated factors control the success of the various procedures that have been advocated; these are the preservation of an intact vascular endothelium and the maintenance of adequate adenine nucleotide reserves. The damaging effect of warm ischemia is ascribed primarily to microcirculatory occlusion by rigid, ATP-depleted red cells, and secondarily to other effects of adenine nucleotide deficiency. It is suggested that continuous perfusion is a valuable technique when there has been significant warm ischemia, because it provides an effective method of restoring a patent microcirculation and is also useful when prolonged storage is required, since adenine nucleotide levels are better maintained than by the available alternatives. However, careful selection of perfusates is necessary to avoid additional perfusion injury to the endothelium. The criteria for effective preservation without continuous perfusion appear to be: little if any warm ischemic injury, effective removal of blood prior to storage, and the inclusion in the washout fluid of relatively impermeant solutes that will prevent cell swelling (particularly endothelial swelling) during preservation. An integrated approach involving both “washout” and perfusion techniques is suggested.     

The assessment of renal preservation by normothermic bloodless perfusion.

A method of estimating renal function by normothermic perfusion in vitro has been developed. In this paper, its application to the study of different methods of hypothermic renal preservation in the rabbit is described. Groups of kidneys were stored at 4 °C for 24 hr by surface cooling alone, by initial perfusion followed by storage (washout perfusion), and by continuous perfusion. Renal function was found to be severely compromised after surface cooling alone or after washout perfusion with an isotonic solution resembling extracellular fluid. Washout with a solution containing sufficient additional glucose to raise the osmolality to 400 mosm/kg gave greatly improved function, but increasing the concentration of magnesium from 2 to 72 mequiv/litre failed to confer any additional benefit, and increasing the concentration of potassium from 4 to 74 mequiv/ litre depressed function. Continuous perfusion with a solution containing albumin and dextran gave results that were inferior to the best washout method, but increasing the osmolality of the perfusate with glucose again resulted in a very significant improvement in function, which however was still inferior to the best washout method of storage. The further use of this test system to study methods of renal preservation is advocated.     

荧光能量转移和偏振研究小牛胸腺DNA与组蛋白在高低离子盐溶液中的离合

本文研究小牛胸腺DNA和组蛋白在体外低、高离子强度盐溶液中的动态缔合与解离。 实验结果是低离子溶液中荧光给体DANsyl-Cl-组蛋白在发射峰位上荧光强度降低,荧光受体吖啶橙-DNA在发射峰位上的荧光强度增高。此两峰位上荧光受体的荧光增量比值是2.7/1(大于1),有能量转移发生,DNA和组蛋白缔合。高离子溶液中两峰位上受体的荧光增量比值降到1.6/1,能量转移减少,DNA和组蛋白解离。 低离子溶液中所得的吖啶橙-DNA的荧光偏振度P值小而高离子溶液中的P值大。说明解离的DNA硷基排列比缔合的DNA硷基排列有序程度强,进一步证明低离子溶液中DNA和组蛋白是缔合的,而高离子溶液中它们是解离的。     

Phosphate Uptake and Root Electrical Potential

It is shown that the potential difference between the rootsof five plant species and their external solution is relatedto the ionic strength of the solution in a logarithmic fashionas predicted by diffuse double-layer theory. The slope of thestraight-line relationship indicates a change of 48 mV for atenfold change in the ionic strength of the solution. At lowphosphate concentrations the rate of uptake is linearly relatedto the square root of the ionic strength of the solution. Thisis in agreement with the effect of ionic strength on surfacepotential and in turn with the effect of potential on surfaceconcentration of adsorbed ions.     

Fixation of neural tissue for electron microscopy with an electronically controlled perfusion pump

Many attempts to improve the perfusion of mammalian tissues aim at changes of the osmotic pressure. We describe a method for fixation of nervous tissues controlling both the hydrostatic pressure and the flow rate of a perfusion solution. The constancy of these parameters is guaranteed by an electronically controlled perfusion pump. Thus, a more uniform and complete preservation can be achieved. Further advantages of this method include provision for a rapid succession of rinsing and fixation solution and a continuous control of the hydrostatic pressure during perfusion.     

Acceleration by montmorillonite of nitrification in soil

A soil naturally containing montmorillonite (M) was amended with 10% M and sequentially perfused with glycine, with fresh glycine being added every 16–17 d after nitrification of the previously added glycine-nitrogen had reached a plateau. In some systems, the old perfusates were replaced each time with a fresh glycine solution; in others, the initial perfusate was not replaced but only adjusted each time to the original 200 ml volume and a comparable glycine concentration (140 μg NH₂-N/ml). The incorporation of M enhanced the rates of heterotrophic degradation of glycine and subsequent autotrophic nitrification, but these stimulatory effects decreased with each successive perfusion. The reasons for these decreases are not known, but they did not appear to be related to inorganic nutrition, as perfusion with a mixed cation solution after five perfusion cycles did not significantly enhance nitrification in either the check or M-amended soils during three subsequent perfusions with glycine. The enhancement of nitrification by M appeared to be a result, in part, of the greater buffering capacity of the M-amended soil, as indicated by lesser reductions in the pH of perfusates from the M-amended soil, by titration curves of the soils, and by the greater and longer stimulation of nitrification in the cheek soil amended with 1% CaCO₃, which had a greater buffering capacity than did M. The stimulation by CaCO₃ may also have been partially the result of providing CO₂ for the autotrophic nitrifyers. Significant concentrations of nitrite accumulated only in perfusates from soil amended with CaCO₃. Air-drying and remoistening the soils enhanced nitrification of subsequently added glycine, especially in the check soil. The importance of pH-mediation, of the production of inhibitors, and/or of feed-back inhibition was indicated by the lower rate and extent of nitrification in systems wherein the perfusates were not replaced between successive additions of glycine. Although the results of these studies confirmed previous observations that M enhances the rate of nitrification in soil, the mechanisms responsible for this stimulation are still not known.     

The capacitance of skeletal muscle fibers in solutions of low ionic strength

The capacitance of skeletal muscle fibers was measured by recording with one microelectrode the voltage produced by a rectangular pulse of current applied with another microelectrode. The ionic strength of the bathing solution was varied by isosmotic replacement of NaCl with sucrose, the [K] [Cl] product being held constant. The capacitance decreased with decreasing ionic strength, reaching a value of some 2 µF/cm² in solutions of 30 mM ionic strength, and not decreasing further in solutions of 15 mM ionic strength. The capacitance of glycerol-treated fibers did not change with ionic strength and was also some 2 µF/cm². It seems likely that lowering the ionic strength reduces the capacitance of the tubular system (defined as the charge stored in the tubular system), and that the 2 µF/cm² which is insensitive to ionic strength is associated with the surface membrane. The tubular system is open to the external solution in low ionic strength solutions since peroxidase is able to diffuse into the lumen of the tubules. Twitches and action potentials were also recorded from fibers in low ionic strength solutions, even though the capacitance of the tubular system was very small in these solutions. This finding can be explained if there is an action potential—like mechanism in the tubular membrane.     

Separation of human serum albumin and human immunoglobulins using carrier phase ultrafiltration

The fractionation of the plasma proteins human serum albumin (HSA) and human immunoglobulins (HIgG) using the combination of two newly developed techniques, pulsed sample injection technique and carrier phase ultrafiltration (CPUF), is discussed in this paper. The effects of pH and ionic strength on the transmission of a single protein (i.e., either HSA or HIgG) through 100 and 300 kDa MWCO polyethersulfone (PES) membranes were quantified using the pulsed sample injection technique. The experimental results thus obtained suggested that it would be possible to fractionate these proteins by optimizing the solution pH and ionic strength. With 100 and 300 kDa PES membranes, effective separation of HSA and HIgG was achieved by CPUF using suitable conditions, i.e., pH 4.7 and low salt concentration. The fractionation of HSA and HIgG by "reverse selectivity" using 300 kDa membranes was also examined.     

Salt-assisted acid hydrolysis of chitosan to oligomers under microwave irradiation

The effect of inorganic salts such as sodium chloride on the hydrolysis of chitosan in a microwave field was investigated. While it is known that microwave heating is a convenient way to obtain a wide range of products of different molecular weights only by changing the reaction time and/or the radiation power, the addition of some inorganic salts was shown to effectively accelerate the degradation of chitosan under microwave irradiation. The molecular weight of the degraded chitosan obtained by microwave irradiation was considerably lower than that obtained by traditional heating. Moreover, the molecular weight of degraded chitosan obtained by microwave irradiation assisted under the conditions of added salt was considerably lower than that obtained by microwave irradiation without added salt. Furthermore, the effect of ionic strength of the added salts was not linked with the change of molecular weight. FTIR spectral analyses demonstrated that a significantly shorter time was required to obtain a satisfactory molecular weight by the microwave irradiation-assisted inorganic salt method than by microwave irradiation without inorganic salts and conventional technology.