Selective N-desulfation of heparin with dimethyl sulfoxide containing water or methanol.

A solvolytic N-desulfation of heparin was developed by treatment of its pyridinium salt with dimethyl sulfoxide containing 5% of water or methanol for 1.5 h at 50 degrees. Chemical and chromatographic studies showed that the solvolytic desulfation is a useful method for N-desulfation of heparin without depolymerization of the heparin molecule. The partially N-desulfated heparins were also obtained by treatment with dimethyl sulfoxide containing 5% of water at 20 degrees, and their anticoagulant activity is related to the degree of N-desulfation.     

Stability of ester sulfates in heparin to solvolysis and dilute acid treatment

The stability of ester sulfates in porcine intestinal heparin to solvolytic desulfation (100 degrees C, 9 H) with dimethylsulfoxide containing 2% pyridine was examined, in comparison with the case of dilute acid treatment (0.1 M HCl, 100 degrees C, 70 min). The resulting heparin modifications were deaminated and the deamination products were fractionated by the procedures reported previously (1978) J. Biochem. 83, 1567-1575). The yields of disulfated disaccharide (b-2) and monosulfated disaccharides (e-2-1 and e-2-3) indicated that 2-O-sulfates in L-iduronic acid residues of heparin were more libile than 6-O-sulfates in glucosamine residues to the dilute acid treatment, whereas the opposite was the case for the solvolysis. The product of heparin modification by solvolysis was similar to whale heparin in the distribution of ester sulfates.     

Preparation of methyl glycosides of di- and higher oligosaccharides from glycosaminoglycuronans by solvolysis with dimethyl sulfoxide containing methanol

Solvolysis of chondroitin 4- or 6-sulfate (pyridinium salt) with dimethyl sulfoxide containing 10% of methanol for 18 h at 95° resulted in the cleavage of the 2-amino-2-deoxy-D-glucoside bonds together with initial desulfation to give methyl β-glycosides of N-acetylchondrosine as a main product and, in addition, higher oligosaccharides, without any loss of uronic acid. Dermatan sulfate was also depolymerized to yield methyl glycosides of di- and higher oligosaccharides under the same conditions. Hyaluronic acid (free acid) was depolymerized by the same solvent in the presence of an equimolar amount of pyridine-sulfur trioxide or pyridinium sulfate per disaccharide unit to give methyl glycosides of di- and higher oligosaccharides. In contrast N-desulfated, N-acetylated heparin was stable under these solvolytic conditions and did not yield heparin oligosaccharides.     

Depolymerization of glycosaminoglycuronans into di- and higher molecular-weight oligo-saccharides: Improved preparation of N-acetyldermosine and oligomeric N-acetylchondrosines

Nonsulfated di- to octadeca-saccharides having 2-acetamido-2-deoxy-d-galactose at the reducing end were prepared, in 81% yield, by treatment of chondroitin 6-sulfate (pyridinium salt) with dimethyl sulfoxide containing 10% of water for 14 h at 90°. N-Acetylchondrosine and N-acetyldermosine were obtained from dermatan sulfate of rooster comb, in 30% and 38% yields, respectively, by solvolysis with dimethyl sulfoxide, containing 10% of water, for 30 h at 105°. Hyaluronic acid was also depolymerized by the same solvent in the presence of an equimolar amount of pyridinium sulfate or chloride per disaccharide unit to give reducing di- and higher molecular weight oligo-saccharides. The results of solvolytic desulfation and depolymerization are compared with those of the conventional methods by acid hydrolysis.     

An improved method for the preparation of chondroitin by solvolytic desulfation of chondroitin sulfates

By heating the pyridinium salts of chondroitin 4- and 6-sulfates in dimethyl sulfoxide containing 10% of water or methanol at 80 degrees C for 1--5 h, several chondroitin preparations with sulfur contents of 0.02--1.05% were prepared in 83--96% yields, respectively. Chemical properties of the preparations, such as degrees of desulfation and of depolymerization, were compared with those of the products obtained by the previously described methods.     

Solvolytic desulfation of 2-deoxy-2-sulfoamino-D-glucose and D-glucose 6-sulfate

Solvolytic desulfation of the pyridinium salts of 2-deoxy-2-sulfoamino-D-glucose and D-glucose 6-sulfate in dimethyl sulfoxide containing 5% of water or methanol was studied to develop a method for selective N-desulfation of heparin. The first-named salt was the most susceptible to N-desulfation.     

(1)H nuclear magnetic resonance spectroscopic analysis for determination of glucuronic and iduronic acids in dermatan sulfate, heparin, and heparan sulfate

(1)H NMR spectroscopy has been established for the determination of uronate residues in glycosaminoglycans (GAGs) such as dermatan sulfate (DS), heparin (HP), and heparan sulfate (HS). Because of variation in the sulfonation positions in DS, HP, or HS, interpretation of spectra is difficult. Solvolysis was applied to remove O-sulfo groups from these GAG chains in dimethyl sulfoxide containing 10% methanol at 80 degrees C for 5 h. In the cases of HP and HS, N-sulfo groups on glucosamine residues were also removed under the same conditions. The resulting unsubstituted amino groups in HP and HS chains were re-N-acetylated using acetic anhydride to obtain homogeneous core structure with the exception of the variation of uronate residues. The contents of glucuronate and iduronate residues in the chemically modified DS, HP, and HS samples were analyzed by 600-MHz (1)H NMR spectroscopy. These methods were applied to compositional analysis of uronate residues in GAGs isolated from various sources.     

Cryoenzymology of penicillopepsin; with an appendix: mechanism of action of aspartyl proteinases

Intrinsic spectral and kinetic properties of penicillopepsin and its action on N-acetylalanylalanyllysyl-p-nitrophenylalanylalanylalanine amide have been investigated at subzero temperatures in aqueous methanol and dimethyl sulfoxide solutions in an attempt to find evidence for or against a covalent mechanism in the catalyzed hydrolysis of peptide bonds. The study of fluorescence and circular dichroism spectra as a function of solvent concentrations gave no evidence for any solvent-induced structural effects at temperatures below the thermal denaturation transition. The effect of temperature on the intrinsic fluorescence of penicillopepsin in either 60% (v/v) methanol or 50% (v/v) dimethyl sulfoxide did not indicate any temperature-induced structural changes. On the other hand, Arrhenius plots for the hydrolysis reaction over the range 0 to -50 degrees C showed downward curvature. A probable explanation for this phenomenon is that the reduction in flexibility of the enzyme due to thermal and viscosity factors leads to the stabilization of a nonproductive conformation. The pH optima of kcat/Km are shifted from 5.1 in aqueous solvents to 5.6 in 60% methanol and to 6.6 in 50% dimethyl sulfoxide. Aqueous methanol caused small decreases of Km and of Kcat; the decrease in the latter was greater than that brought about by the decrease in the water concentration. In aqueous dimethyl sulfoxide, there was no detectable change in kcat up to 15%, but Km increased by more than an order of magnitude. Above 15%, only kcat/Km could be measured. No evidence for the accumulation of either covalent amino or covalent acyl intermediates was obtained when penicillopepsin was incubated at -70 degrees C in 67% methanol with several substrates. Although negative, these experiments do not rule out conclusively the involvement of covalent intermediates in penicillopepsin-catalyzed reactions.     

The effect of cryopreservation on ciliary beat frequency of human respiratory epithelium

The effect of cryopreservation on ciliary activity of human nasal respiratory epithelium was evaluated. Samples were cryopreserved in a solution containing nutrient medium, 10% fetal calf serum, and two different concentrations (10 or 20%) of dimethyl sulfoxide and stored in liquid nitrogen at -196 degrees C for 2 weeks. Ciliary beat frequencies (CBF) of the samples before and after cryopreservation were compared. Mean CBF values did not differ significantly with both concentrations of dimethyl sulfoxide. The mean intrasample coefficient of variation of the CBF decreased significantly after cryopreservation. After thawing, CBF remained unchanged for at least 4 hr. It is concluded that normal ciliated epithelial cells can be frozen and stored in liquid nitrogen at 196 degrees C while maintaining their CBF.     

Effect of sample transport systems on survival of bacteria in ground beef.

The effects of two transport systems and cryoprotective agents on the survival of bacteria in ground beef samples were evaluated. Survival of Clostridium perfringens in ground beef samples after simulated transport (72 h) was higher (about 99%) in Dry Ice than in Trans Temp shipping units (-3 degrees C). There were no significant differences between the two transport systems in survival of coliforms, Escherichia coli, Staphylococcus aureus, or aerobic bacteria. Mixing ground beef samples at a ratio of 1:1 (wt/vol) with 10, 20, or 30% buffered solutions of dimethyl sulfoxide or glycerol before freezing improved the survival of C. perfringens and coliforms in both transport systems. Recovery of E. coli was significantly higher with the addition of 10% dimethyl sulfoxide before Dry Ice transport. Addition of 10% dimethyl sulfoxide resulted in a 100% recovery of both S. aureus and aerobic bacteria from ground beef after simulated transport in Trans Temp shipping units. The use of cryoprotective agents can improve the survival of bacteria during transport of ground beef samples.     

Effect of sample transport systems on survival of bacteria in ground beef.

The effects of two transport systems and cryoprotective agents on the survival of bacteria in ground beef samples were evaluated. Survival of Clostridium perfringens in ground beef samples after simulated transport (72 h) was higher (about 99%) in Dry Ice than in Trans Temp shipping units (-3 degrees C). There were no significant differences between the two transport systems in survival of coliforms, Escherichia coli, Staphylococcus aureus, or aerobic bacteria. Mixing ground beef samples at a ratio of 1:1 (wt/vol) with 10, 20, or 30% buffered solutions of dimethyl sulfoxide or glycerol before freezing improved the survival of C. perfringens and coliforms in both transport systems. Recovery of E. coli was significantly higher with the addition of 10% dimethyl sulfoxide before Dry Ice transport. Addition of 10% dimethyl sulfoxide resulted in a 100% recovery of both S. aureus and aerobic bacteria from ground beef after simulated transport in Trans Temp shipping units. The use of cryoprotective agents can improve the survival of bacteria during transport of ground beef samples.     

Influence of extender, cryoperservative and seminal processing procedures on postthaw motility of canine spermatozoa frozen in straws

Experiments were conducted to evaluate two extenders (egg-yolk Tris and egg-yolk lactose), varying concentrations of two cryopreservatives (glycerol and dimethyl sulfoxide), and rates for cooling to 5 degrees C, cooling from 5 to -100 degrees C, and warming for canine spermatozoa packaged in 0.5-ml French straws. At optimal concentrations of glycerol, egg-yolk Tris extender was superior to egg-yolk lactose in preserving spermatozoal motility. Addition of dimethyl sulfoxide, alone or in combination with glycerol in either extender, was not beneficial to spermatozoal survival after thawing. Canine spermatozoa withstood a range of cooling and equilibration times with no detrimental effect on spermatozoal motility prior to freezing. However, there were differences in spermatozoal motility immediately after thawing; these differences were variable, resulting in a cooling time by equilibration time interaction. Spermatozoal motility after thawing was best preserved by freezing in egg-yolk Tris extender containing 2-4% glycerol, using a moderate rate of cooling from 5 to -100 degrees C (-5 degrees C/min from 5 to -15 degrees C, then -20 degrees C/min from -15 to -100 degrees C). Three of 12 bitches inseminated intravaginally with semen frozen using this protocol became pregnant.     

Survival of mint shoot tips after exposure to cryoprotectant solution components

Many plant species can be cryopreserved by treating shoot tips with complex cryoprotectant solutions before rapidly cooling them to liquid nitrogen temperatures. Plant vitrification solution 2 (PVS2), a commonly selected cryoprotectant, can be lethal with extended exposure times. To determine potentially toxic combinations, we have exposed mint shoot tips to one-, two-, three-, and four-component solutions of PVS2 chemicals (30% glycerol, 15% ethylene glycol, 15% dimethyl sulfoxide, and 0.4 M sucrose) at 0 and 22 degrees C. Overall, solution exposures at 22 degrees C were more damaging than exposures at 0 degree C. Solutions with glycerol, particularly in combination with ethylene glycol and dimethyl sulfoxide, were also damaging. Cryoprotectant solutions PGluD (10% PEG8000, 10% glucose, and 10% dimethyl sulfoxide) and PVS3 (50% glycerol, 50% sucrose) were less damaging than PVS2 at 22 degrees C. When plant cryoprotectants are characterized on a toxicological and biophysical basis, less damaging cryoprotectant solutions could be developed.     

The hydrolytic cycle of sarcoplasmic reticulum Ca2+-ATPase in the absence of calcium

The hydrolytic cycle of sarcoplasmic reticulum Ca2+-ATPase in the absence of Ca2+ was studied. At pH 6.0, 10 degrees C and in the absence of K+, the enzyme displays a very low velocity of ATP hydrolysis. Addition of up to 15% dimethyl sulfoxide increased this velocity severalfold (from 5-18 nmol of Pi X mg of protein-1 X h-1) and then decreased at higher solvent concentrations. Dimethyl sulfoxide increased both enzyme phosphorylation from ATP and the affinity for this substrate. Maximal levels of 1.0-1.2 nmol of EP X mg of protein-1 and apparent KM for ATP of 5 X 10(-6) M were obtained at a concentration of 30% dimethyl sulfoxide. The same preparation under optimal conditions (pH 7.5, 10 microM CaCl2, 100 mM KCl and no dimethyl sulfoxide at 37 degrees C) displays a velocity of ATP hydrolysis between 8 and 12 X 10(5) nmol of Pi X mg of protein-1 X h-1 while the phosphoenzyme levels varied between 3.5 and 4.0 nmol of EP X mg of protein-1. Enzyme phosphorylation from ATP in the absence of Ca2+ always preceded Pi liberation into the assay media. Two different phosphoenzyme species were formed which were kinetically distinguished by their decomposition rates. The observed steady-state velocity of ATP hydrolysis could be accounted for either by the decay of the fast component or by the simultaneous decomposition of both phosphoenzyme species. The hydrolysis of the phosphoenzyme formed in the absence of Ca2+ was KCl-stimulated and ADP-independent. The rate constant of breakdown was equal to that observed for the phosphoenzyme formed in the presence of Ca2+. It is suggested that the rapidly decaying phosphoenzyme (and possibly both rapidly and slowly decaying species) are intermediates in the reaction cycle of Mg2+-dependent ATP hydrolysis of sarcoplasmic reticulum Ca2+-ATPase and may represent a bypass of Ca2+ activation by dimethyl sulfoxide.     

Loss of positional specificity in the aminoacylation of Escherichia coli tRNAGly

The positional specificity in the aminoacylation of Escherichia coli tRNAGly by its cognate aminoacyl-tRNA synthetase has been studied using tRNAGlys terminating in 2'- or 3'-deoxyadenosine under conditions believed to alter tRNA conformation. Although E. coli tRNAGly terminating in 3'-deoxyadenosine has been reported not to be a good substrate for activation by the homologous glycyl-tRNA synthetase, by systematic variation of the conditions employed for aminoacylation it was possible to activate this tRNA to essentially the same extent as unmodified tRNAGly. Activation of tRNAGly terminating in 3'-deoxyadenosine was carried out optimally at 45 degrees C in an incubation mixture containing 0.3-0.4 M NaCl; 10% methanol, ethanol, and dimethyl sulfoxide were found to facilitate activation of the modified tRNA. Interestingly, the conditions employed to enhance activation of this modified tRNAGly had no effect on the activation of unmodified tRNAGly or tRNAGly terminating in 2'-deoxyadenosine. These experiments afford insight into the activation of tRNAGly by glycyl-tRNA synthetase and provide facile access to positionally defined, isomeric glycl-tRNAGlys.     

Comparison of different extenders for the cryopreservation of Atlantic salmon spermatozoa

Fifteen extenders were produced by adding dimethyl sulfoxide (DMSO) at 8, 10 or 12% of diluent volume to 5 diluents. All extenders were cooled to 4 degrees C. Pooled Atlantic salmon (Salmo salar ) semen with greater than 90% progressive motility was kept at 4 degrees C and added to each extender so that the semen was diluted 1:3 (semen:extender). The equilibration time was less than 5 minutes at 4 degrees C. The extended semen was loaded into 0.5-ml straws and was cooled from 4 degrees C to -90 degrees C at a rate of 30 degrees C per minute. The straws were then plunged into liquid nitrogen for storage. Fluorometry was used to determine the viability of the semen in each of the extenders after freezing and thawing. Cryopreservation of Atlantic salmon semen in Extender 3 (0.137 M NaCl, 0.011 M KCl, 0.004 M Na(2)HPO(4).7H(2)O, 7.5 g/l L-alpha-lecithin and 12% dimethyl sulfoxide) and Extender 12 (0.100 M KHCO(3), 0.0065 M reduced glutathione, 0.125 M sucrose and 12% dimethyl sulfoxide) resulted in significantly (P<0.05) lower percentages of dead spermatozoa than for the other extenders. Furthermore, there was a significantly (P<0.05) lower percentage of dead cells in Extender 3 than in Extender 12.     

Preliminary experiments on the cryopreservation of penaeid shrimp (Penaeus japonicus) embryos, nauplii and zoea

To improve availability of penaeid seedstock during periods of high demand, experiments were conducted to determine the feasibility of stockpiling embryos by freezing them. Embryos were screened for developmental stage; cryoprotectants, chilling effects, and freezing regimens were likewise evaluated. Juvenile forms (embryos, nauplii and zoea) of Penaeus japonicus were exposed to various cryoprotectants, including dimethyl sulfoxide, glycerol, methanol, ethylene glycerol and polyethylene glycol 300 under ambient temperature (25 degrees C). Following this bioassay, maximum safe concentrations of each cryoprotectant were tested on the juveniles under chilling to 0 degree C and with 42 freezing regimens. Methanol was found to be relatively nontoxic. Early developmental stages were the most sensitive to chilling. Initial attempts to freeze P. japonicus juveniles were reported. The survival rate of nauplii and zoea treated with 10% methanol in natural sea water (35 ppt salinity) and frozen to -15 degrees C was 85%, and some nauplii and zoea survived freezing to -25 and -196 degrees C. However, no treatment yielded normal nauplii or zoea after freezing.     

The effects of dimethyl sulfoxide on the kinetics of tubulin assembly

The kinetics of tubulin assembly were examined in the absence and presence of dimethyl sulfoxide at 37 degrees C. Inclusion of 1.4 M (10%) dimethyl sulfoxide lowered the critical protein concentration about 8-10-fold, from 9.4 microM in the absence of the organic solvent to 1.1 microM in its presence. This decrease was due solely to an effect on k-, the off rate constant. The on rate constant k+, was essentially unaffected. Another effect of dimethyl sulfoxide was in the nucleation process. The pseudo-first-order rate constant of elongation, kapp (k+[m]), was greatly increased by inclusion of dimethyl sulfoxide. This was due to an increase in the microtubule number concentration, [m]. The microtubules formed in the presence of dimethyl sulfoxide were much shorter than those formed in its absence, accounting for the higher number concentration. The nucleation number, n, was calculated by plots of ln kapp vs. ln c0 or ln t10% vs. ln c0, and the value appeared to be about 4 to 5, although some variability was found. It was shown that a plot of kapp vs. c0 to determine n, is not appropriate because of the inability to distinguish between linear and curved plots in the range of tubulin concentration used in assembly studies.     

Optimization of cryoprotectants for cryopreservation of rat hepatocyte

Rat hepatocytes were cryopreserved in hormonally-defined medium (HDM) containing either fetal bovine serum (FBS), glycerol, dimethyl sulfoxide (DMSO), sucrose or a mixture of these as a cryoprotectant. The best survival was with 10% (v/v) DMSO containing 30% (v/v) FBS using 5 x 10(5) hepatocytes ml(-1) at -70 degrees C for 5 d on type I collagen-coated dishes. After thawing, the cell viability was 81% determined by the MTT-test. The cryopreserved hepatocytes had the capacity of albumin synthesis similar to hepatocytes without cryopreservation. This result shows that cryopreservation of rat hepatocyte can be used for the evaluation of hepatic functions.     

Purification, biochemical and molecular characterization of a metalloprotease from Pseudomonas aeruginosa MN7 grown on shrimp wastes

A protease-producing bacterium was isolated and identified as Pseudomonas aeruginosa MN7. The strain was found to produce proteases when it was grown in media containing only shrimp waste powder (SWP), indicating that it can obtain its carbon, nitrogen, and salts requirements directly from shrimp waste. The use of 60 g/l SWP resulted in a high protease production. Elastase, the major protease produced by P. aeruginosa MN7, was purified from the culture supernatant to homogeneity using acetone precipitation, Sephadex G-75 gel filtration, and ultrafiltration using a 10-kDa cut-off membrane, with a 5.2-fold increase in specific activity and 38.4% recovery. The molecular weight of the purified elastase was estimated to be 34 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. The optimum temperature and pH for protease activity were 60 degrees C and 8.0, respectively. The activity of the enzyme was totally lost in the presence of ethylene glycol tetraacetic acid, suggesting that the purified enzyme is a metalloprotease. The purified enzyme was highly stable in the presence of organic solvents, retaining 100% of its initial activity after 60 days of incubation at 30 degrees C in the presence of dimethyl sulfoxide and methanol. The lasB gene, encoding the MN7 elastase, was isolated and its DNA sequence was determined.