- Email: [email protected]
Venous allograft preservation (preliminary results)
586
ABSTRACTS,
25th ANNUAL
ulate membrane fluidity, based on selective reduction of cis double bonds of esteritied fatty acids with a water-soluble hydrogenation catalyst, the Pd(I1) complex of alizarin red, Pd(QS),. i Application of this technique permits a direct evaluation of the adaptive role of lipid saturation in the absence of temperature change. In the present paper two areas are highlighted where the hydrogenation technique was exploited. Complementary biophysical techniques have provided evidence for effects of programmed reduction of double bond content on lipid fluidity/molecular order/ phase behavior in isolated chloroplast. Progressive saturation of double bonds inhibited first the electron transport between the photosystems, folowed by the inhibition of electron flow around PSII. The hypothesis that the level of microviscosity regulates the electron flow via controlling the rate of diffusion of reduced plastoquinone pool has not been confirmed experimentally. Examples of hydrogenation conduced on living cells and protoplasts under conditions permitting full recovery are also discussed. 228. Alteration of Penicillium Expansum through Lyophilization and Cryopreservation. G. L. HENNEBERT AND J. F. BERNY (Mycotheque de I’Universitt Catholique de Louvain, Louvain-la-Neuve, Belgium). Sixteen preservation procedures are applied to monoconidial strain of Penicillium expansum MUCL in order to test the strain stability in regard to its macroscopic and microscopic characteristics. The six procedures of lyophilisation are freeze-drying in one step, centrifugal freeze-drying in two steps, and shelf freeze-drying in two steps at 1 and SO”C/min freezing rate and at slow and fast warming rate. Cryopreservation is applied after freezing at 1 and SO”C/min and storage at 20, 40, 80, and 135°C in electrical freezers and at 196°C in liquid nitrogen. The procedures are carried out by three distinct culture collections, IMI, CBS, and IPM. Storage at -20°C after slow freezing and storage in liquid nitrogen are duplicated. Skim milk (10%) is used in lyophilization and glycerol (10%) in cryopreservation. The strain revived immediately after preservation as well as the untreated strain have been investigated within 3 months in polyconidial cultures (5000 conidia) and monoconidial cultures (200 single conidia) on six different culture media. Sectorization of the polyconidial colonies is observed after most of the procedures with exception of after centrifugal freeze drying (at CMI) and after cryopreservation at -30 (at IPM) and at - 135°C. numerous sectors have been observed at CMI, and some occasional at sectors CBS. Sectors have been proven to result from the alteration of strains characteristics such as length i The Pd(QS), catalyst is available from Molecular Probes, Eugene, OR.
MEETING
of conidiophore, coremial formation, color of conidia, and growth speed in the inoculated conidia. Indeed, cultures from single conidia of the same origin have displayed nine types of variation differing from the typical coremial strain by a combination of the variating characteristics. The amount of variant conidia from the first conidial generation after treatment is as much as 76%. The conidia observed after cryopreservation at - 80 and - 135°C are typical and similar in all respects to those of the untreated original strain. Studies of the character stability of the nine selected variant strains are carried out in successive generation by monoconidial transfer. SESSION
XIX-SELECTED
TOPICS
229. Iron Release and Free Radical Damage to Rabbit Kidneys Subjected to Cold Ischemia. J. D. GOWER, G. HEALING, AND C. J. GREEN (Surgical
B. J. FULLER,*
Research Group, Clinical Research Centre, Watford Road, Harrow, Middlesex, United Kingdom, and *Academic Department of Surgery, Royal Free Hospital School of Medicine, Pond Street, London, United Kingdom). We have previously demonstrated that rabbit kidneys subjected to periods of cold storage at 0°C exhibit significantly elevated rates of lipid peroxidation upon subsequent homogenization and incubation at 37°C. Lipid peroxidation is initiated by oxygen-derived free radicals, the hydroxyl radical (OH) being the most damaging species. OH’ production is dependent on the presence of transition metals and a small pool of catalytic iron is present intracellulary in the form of low molecular weight chelates. We have developed a method for the determination of chelatable iron in kidney homogenates which involves the HPLC analysis of iron complexes with desfenioxamine. Using this method it was found that the level of chelatable iron was significantly elevated to double control levels in both the cortex and medulla of kidneys following flush and 24 hr storage at 0°C in hypertonic citrate solution. No difference in the total iron content of the organs was observed. This redistribution of iron during storage is likely to be the result of iron removal from the storage protein ferritin facilitated by the reducing environment in the ischernic tissue. An increase in the intracellular level of catalytic iron may therefore be an important factor in the mechanism of increased oxidative stress to the tissues following periods of ischemia. 230. Venous Allograft Preservation (Preliminary Results). F. B~HM, P. ZDRAHAL, M. VRANA, A. SEICHERTOVA,
AND
V. RUZBAR~KP
tute of Clinical and Experimental Prague, Czechoslovakia).
(Insti-
Medicine,
The two main problems seen as a priority in current
ABSTRACTS,
25th ANNUAL
research of venous allotransplantation are efficient long-term preservation and management of posttransplantation immunologic complications. In the series of experiments summarized in this study, we used the most common method for freezing and immunosuppression with Cyclosporin A (CyA). The external jugular veins of mongrel dogs (weighing 25-30 kg) were removed and used to replace right common carotid arteries. The high perfusion flow through the graft, instrumental in maintaining its patency, was reduced to 80 mYmin. The freezing medium was homologous serum or deactivated bovine serum with lO-15% of Me,SO. Venous grafts were placed right into the medium previously cooled in an ice-water bath. The cooling rate was a mean 0.93”C/min (SD 0.24) to phase change, then -S’C/min to -8O”C, and then to LN2. The rewarming rate was about 4OO”C/min in 40°C water bath. The grafts were then transplanted without Me,SO washout. CyA was administered orally at a dose of 20 m&g/day for 6 weeks or added to the freezing medium at a single dose of 50 mg/liter. Recipient blood CyA levels ranged between 200 and 400 &liter. Histology and electron microscopy showed that this method for cryopreservation did not confer absolute protection to endothelial cells. Venous allotransplants remain patent for 6 weeks in 100% of cases (five out of five), and two out of five for more than 24 weeks once CyA administration has been discontinued. In our experiments, patency of the vessel depended on its localization (ipsilaterally right to the right side, four out of five occluded following contralateral transplantation from the right to the left side) and not on the preservation time at -196°C (12-134 days). A 70% patency rate was achieved in a control group following autotransplantation of cryopreserved vessels, but the sequence of right and left vein removal and transplantation was not registered. Because both right and left veins were removed one after another, but frozen together during a single procedure, further experiments will be needed to identify differences in treatment that exert an effect on the outcome of cryopreserved vein autotransplantation. Addition of CyA to the freezing
587
MEETING
medium had no positive effect on maintaining transplant patency.
allo-
231. Freezing of Rabbit Embryos in the Presence of Methanol.
K. PAPIS AND J. DZIAK
(Institute
of
Genetics and Animal Breeding, Jastrzebiec, 05551 Mrokow, Poland). It has been shown previously that mouse embryos may survive deep freezing in the presence of 3.0 M methanol if they are slowly frozen to -40°C before they are plunged into liquid nitrogen and transferred into recipients without dilution of methanol. We have examined the ability of rabbit morulae, frozen under similar conditions, to survive in vitro and in vivo. Embryos were recovered in polybenzimidazole (PBI) solution from superovulated donors 65 h after mating. Morulae were plunged directly into 3.0 A4 methanol and then placed into test tubes or plastic straws. After seeding at - lO”C, samples were cooled at the rate of l”C/min to - 35°C and then plunged directly into liquid nitrogen. Thawing was performed in a water bath at the rate of 600-lOOO”C/min. Out of 143 embryos frozen in test tubes and 33 frozen in plastic straws 75 (52.4%) and 24 (72.4%), respectively, developed in vitro to the expanded blastocyst stage. Out of 51 additional embryos frozen-thawed in straws, 5 were discarded because of damaged mucin coat. The remaining 46 were transferred without dilution of methanol into the uterus of 8 recipients and resulted in 5 pregnancies, 16 implantations, and I3 (28.3%) viable fetuses. Control transfer of 51 unfrozen embryos equilibrated and transferred in PBI containing 3.0 M methanol resulted in 37 (72.5%) viable fetuses. The results presented suggest low toxicity of 3.0 M methanol to both the rabbit embryos and uterus and confvm a possibility of using methanol as a suitable cryoprotective agent during freezing of rabbit embryos. ARTHUR
W.
ROWE,
EDITOR
New York University Medical Center New York, New York 10016
ABSTRACTS,
25th ANNUAL
ulate membrane fluidity, based on selective reduction of cis double bonds of esteritied fatty acids with a water-soluble hydrogenation catalyst, the Pd(I1) complex of alizarin red, Pd(QS),. i Application of this technique permits a direct evaluation of the adaptive role of lipid saturation in the absence of temperature change. In the present paper two areas are highlighted where the hydrogenation technique was exploited. Complementary biophysical techniques have provided evidence for effects of programmed reduction of double bond content on lipid fluidity/molecular order/ phase behavior in isolated chloroplast. Progressive saturation of double bonds inhibited first the electron transport between the photosystems, folowed by the inhibition of electron flow around PSII. The hypothesis that the level of microviscosity regulates the electron flow via controlling the rate of diffusion of reduced plastoquinone pool has not been confirmed experimentally. Examples of hydrogenation conduced on living cells and protoplasts under conditions permitting full recovery are also discussed. 228. Alteration of Penicillium Expansum through Lyophilization and Cryopreservation. G. L. HENNEBERT AND J. F. BERNY (Mycotheque de I’Universitt Catholique de Louvain, Louvain-la-Neuve, Belgium). Sixteen preservation procedures are applied to monoconidial strain of Penicillium expansum MUCL in order to test the strain stability in regard to its macroscopic and microscopic characteristics. The six procedures of lyophilisation are freeze-drying in one step, centrifugal freeze-drying in two steps, and shelf freeze-drying in two steps at 1 and SO”C/min freezing rate and at slow and fast warming rate. Cryopreservation is applied after freezing at 1 and SO”C/min and storage at 20, 40, 80, and 135°C in electrical freezers and at 196°C in liquid nitrogen. The procedures are carried out by three distinct culture collections, IMI, CBS, and IPM. Storage at -20°C after slow freezing and storage in liquid nitrogen are duplicated. Skim milk (10%) is used in lyophilization and glycerol (10%) in cryopreservation. The strain revived immediately after preservation as well as the untreated strain have been investigated within 3 months in polyconidial cultures (5000 conidia) and monoconidial cultures (200 single conidia) on six different culture media. Sectorization of the polyconidial colonies is observed after most of the procedures with exception of after centrifugal freeze drying (at CMI) and after cryopreservation at -30 (at IPM) and at - 135°C. numerous sectors have been observed at CMI, and some occasional at sectors CBS. Sectors have been proven to result from the alteration of strains characteristics such as length i The Pd(QS), catalyst is available from Molecular Probes, Eugene, OR.
MEETING
of conidiophore, coremial formation, color of conidia, and growth speed in the inoculated conidia. Indeed, cultures from single conidia of the same origin have displayed nine types of variation differing from the typical coremial strain by a combination of the variating characteristics. The amount of variant conidia from the first conidial generation after treatment is as much as 76%. The conidia observed after cryopreservation at - 80 and - 135°C are typical and similar in all respects to those of the untreated original strain. Studies of the character stability of the nine selected variant strains are carried out in successive generation by monoconidial transfer. SESSION
XIX-SELECTED
TOPICS
229. Iron Release and Free Radical Damage to Rabbit Kidneys Subjected to Cold Ischemia. J. D. GOWER, G. HEALING, AND C. J. GREEN (Surgical
B. J. FULLER,*
Research Group, Clinical Research Centre, Watford Road, Harrow, Middlesex, United Kingdom, and *Academic Department of Surgery, Royal Free Hospital School of Medicine, Pond Street, London, United Kingdom). We have previously demonstrated that rabbit kidneys subjected to periods of cold storage at 0°C exhibit significantly elevated rates of lipid peroxidation upon subsequent homogenization and incubation at 37°C. Lipid peroxidation is initiated by oxygen-derived free radicals, the hydroxyl radical (OH) being the most damaging species. OH’ production is dependent on the presence of transition metals and a small pool of catalytic iron is present intracellulary in the form of low molecular weight chelates. We have developed a method for the determination of chelatable iron in kidney homogenates which involves the HPLC analysis of iron complexes with desfenioxamine. Using this method it was found that the level of chelatable iron was significantly elevated to double control levels in both the cortex and medulla of kidneys following flush and 24 hr storage at 0°C in hypertonic citrate solution. No difference in the total iron content of the organs was observed. This redistribution of iron during storage is likely to be the result of iron removal from the storage protein ferritin facilitated by the reducing environment in the ischernic tissue. An increase in the intracellular level of catalytic iron may therefore be an important factor in the mechanism of increased oxidative stress to the tissues following periods of ischemia. 230. Venous Allograft Preservation (Preliminary Results). F. B~HM, P. ZDRAHAL, M. VRANA, A. SEICHERTOVA,
AND
V. RUZBAR~KP
tute of Clinical and Experimental Prague, Czechoslovakia).
(Insti-
Medicine,
The two main problems seen as a priority in current
ABSTRACTS,
25th ANNUAL
research of venous allotransplantation are efficient long-term preservation and management of posttransplantation immunologic complications. In the series of experiments summarized in this study, we used the most common method for freezing and immunosuppression with Cyclosporin A (CyA). The external jugular veins of mongrel dogs (weighing 25-30 kg) were removed and used to replace right common carotid arteries. The high perfusion flow through the graft, instrumental in maintaining its patency, was reduced to 80 mYmin. The freezing medium was homologous serum or deactivated bovine serum with lO-15% of Me,SO. Venous grafts were placed right into the medium previously cooled in an ice-water bath. The cooling rate was a mean 0.93”C/min (SD 0.24) to phase change, then -S’C/min to -8O”C, and then to LN2. The rewarming rate was about 4OO”C/min in 40°C water bath. The grafts were then transplanted without Me,SO washout. CyA was administered orally at a dose of 20 m&g/day for 6 weeks or added to the freezing medium at a single dose of 50 mg/liter. Recipient blood CyA levels ranged between 200 and 400 &liter. Histology and electron microscopy showed that this method for cryopreservation did not confer absolute protection to endothelial cells. Venous allotransplants remain patent for 6 weeks in 100% of cases (five out of five), and two out of five for more than 24 weeks once CyA administration has been discontinued. In our experiments, patency of the vessel depended on its localization (ipsilaterally right to the right side, four out of five occluded following contralateral transplantation from the right to the left side) and not on the preservation time at -196°C (12-134 days). A 70% patency rate was achieved in a control group following autotransplantation of cryopreserved vessels, but the sequence of right and left vein removal and transplantation was not registered. Because both right and left veins were removed one after another, but frozen together during a single procedure, further experiments will be needed to identify differences in treatment that exert an effect on the outcome of cryopreserved vein autotransplantation. Addition of CyA to the freezing
587
MEETING
medium had no positive effect on maintaining transplant patency.
allo-
231. Freezing of Rabbit Embryos in the Presence of Methanol.
K. PAPIS AND J. DZIAK
(Institute
of
Genetics and Animal Breeding, Jastrzebiec, 05551 Mrokow, Poland). It has been shown previously that mouse embryos may survive deep freezing in the presence of 3.0 M methanol if they are slowly frozen to -40°C before they are plunged into liquid nitrogen and transferred into recipients without dilution of methanol. We have examined the ability of rabbit morulae, frozen under similar conditions, to survive in vitro and in vivo. Embryos were recovered in polybenzimidazole (PBI) solution from superovulated donors 65 h after mating. Morulae were plunged directly into 3.0 A4 methanol and then placed into test tubes or plastic straws. After seeding at - lO”C, samples were cooled at the rate of l”C/min to - 35°C and then plunged directly into liquid nitrogen. Thawing was performed in a water bath at the rate of 600-lOOO”C/min. Out of 143 embryos frozen in test tubes and 33 frozen in plastic straws 75 (52.4%) and 24 (72.4%), respectively, developed in vitro to the expanded blastocyst stage. Out of 51 additional embryos frozen-thawed in straws, 5 were discarded because of damaged mucin coat. The remaining 46 were transferred without dilution of methanol into the uterus of 8 recipients and resulted in 5 pregnancies, 16 implantations, and I3 (28.3%) viable fetuses. Control transfer of 51 unfrozen embryos equilibrated and transferred in PBI containing 3.0 M methanol resulted in 37 (72.5%) viable fetuses. The results presented suggest low toxicity of 3.0 M methanol to both the rabbit embryos and uterus and confvm a possibility of using methanol as a suitable cryoprotective agent during freezing of rabbit embryos. ARTHUR
W.
ROWE,
EDITOR
New York University Medical Center New York, New York 10016