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PubMed1998

High-pressure freezing causes structural alterations in phospholipid model membranes.

The influence of high-pressure freezing (HPF) on the lipid arrangement in phospholipid model membranes has been investigated. Liposomes consisting of pure dipalmitoyl-phosphatidylcholine (DPPC) and of DPPC mixed with a branched-chain phosphocholine (1,2-di(4-dodecyl-palmitoyl)- sn-glycero-3-phosphocholine) have been analysed by freeze-fracture electron microscopy. The liposomes were frozen either by plunging into liquid propane or by HPF. The characteristic macroripple-phase of the two-component liposome system is drastically changed in its morphology when frozen under high-pressure conditions. The influence of ethanol which acts as pressure transfer medium was ruled out by control experiments. In contrast, no high-pressure alterations of the pure DPPC bilayer membrane have been observed. We assume that the modification of the binary system is due to a pressure-induced relaxation of a stressed and unstable lipid molecule packing configuration. HPF was performed with a newly designed sample holder, for using sandwiched copper platelets with the high-pressure freezing machine Balzers HPM010. The sandwich construction turned out to be superior to the original holder system with regard to freeze-fracturing of fluid samples. By inserting a spacer between the supports samples with a thickness of 20-100 microns can be high-pressure frozen. The sandwich holder is provided with a thermocouple to monitor cooling rates and allows exact sample temperature control. Despite a two-fold mass reduction compared to the original holder no HPF cooling rate improvement has been achieved (4000 degrees Cs-1). We conclude that the cooling process in high-pressure freezing is determined mainly by cryogen velocity.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1995

Immunolocalization of freezing-tolerance-associated proteins in the cytoplasm and nucleoplasm of wheat crown tissues.

A protein family associated with the development of freezing tolerance in wheat has been identified. This protein family is Gramineae-specific and coordinately regulated by low temperature. Antibodies directed against the 50 kDa (WCS120) protein recognize at least 5 members of this family. Using these antibodies, the cellular content and location of this protein family was determined in cold-acclimated wheat seedlings. Western analyses of subcellular fractions indicated the presence of all members of the family in the cytosolic and purified nuclear fractions. These proteins accumulated to 0.9% of soluble proteins after 21 days of cold acclimation in winter wheat. This represents a cellular concentration of 1.34 microM. Immunohistochemical localization showed that these proteins are highly expressed in the vascular transition zone. No detectable expression was found in mature xylem, in the shoot apical meristem or lateral root primordia. This differential tissue expression suggests that the sensitive cells near the regions where water tends to freeze first require a higher amount of these proteins. This observation is consistent with the fact that regrowth after freezing stress is highly dependent on the viability of this region of the crown. Electron microscopy analysis using immunogold labelling showed that these proteins are present in the cytoplasm and in the nucleoplasm. They are not found in cell walls or other organelles. In vitro cryoprotective assays indicated that the WCS120 protein (PD50 of 10 micrograms ml-1 or 0.2 microM) are as effective as BSA and sucrose (at 250 mM) against freezing denaturation of lactate dehydrogenase. These results suggest that this protein family may be involved in a general mechanism of protection in the soluble fraction of the cell. Their presence in the nucleoplasm may also suggest a possible protective function of the transcriptional machinery. The high hydrophilicity, the abundance and stability of these proteins to boiling suggest that they may provide a particular micro-environment needed for cell survival in the sensitive vascular transition zone during freezing stress.

Antibodies↗
PubMed1999

Preservation of pathological tissue specimens by freeze-drying for immunohistochemical staining and various molecular biological analyses.

Conditions of preserving DNA, RNA and protein in pathological specimens are of great importance as degradation of such macromolecules would critically affect results of molecular biological analysis. The feasibility of freeze-drying as a means of preserving pathological tissue samples for molecular analysis has previously been shown. In the present study, further tests on long-term storage conditions and analyses of freeze-dried samples by polymerase chain reaction (PCR), reverse transcriptase (RT)-PCR, western blotting and immunohistochemistry are reported. Rat chromosomal DNA of freeze-dried samples stored for 4 years showed slight degradation while RNA degradation was more prominently seen at an earlier stage of storage. However, these 4 year DNA and RNA samples were still able to serve as a template for some PCR and RT-PCR analyses, respectively. Overexpression of c-erbB-2 and p53 protein was demonstrated by western blotting and immunohistochemical staining using freeze-dried human breast cancer tissues. Although macromolecules in freeze-dried samples degrade to some extent during the preservation period, they should still be of value for certain molecular biological analyses and morphological examination; hence, providing more convenient and inexpensive ways of pathological tissue storage.

Animals↗
PubMed1980

Cryopreservation of rainbow trout sperm by deep-freezing.

Cryopreservation trials on rainbow trout (Salmo gairdneri) sperm were carried out using two basic extenders: Mounib's medium (M) and Ménézo's medium (Me) to which were added bovine serum albumin (BSA) and tellurite egg yolk (Institut Pasteur). After 10 p. 100 of DMSO was added to these different deep-freeze diluents (DD), they were mixed with the sperm and then deep-frozen into 100 microliter pellets on dry ice. The pellets were stored in liquid nitrogen for periods lasting from 3 days to 6 months. The intensity of sperm motility and fertilizing ability were measured before and after cryopreservation. After the sperm was diluted in Ménézo's medium, slight spermatozoon motility was noticed, which probably caused their early exhaustion and would explain the lower fertilizing ability observed after thawing. Mounib's medium gave better results, especially after 10 p. 100 of egg yolk was added. The optimal deep-freeze conditions were: 1/3 dilution, no equilibration after dilution but immediate deep-freezing at a rate of 10 to 40 degrees C/min. Thawing had to be carried out rapidly in 10 sec. However, the spermatozoa were altered during the freezing-thawing process, and during insemination more frozen spermatozoa had to be used to equal the fertilization rate obtained with non-frozen sperm. However, the fertile spermatozoa gave normal embryogenesis and no abnormal development was seen up to the vesicle resorption stage. At the end of spermiation, sperm fitness for deep-freezing decreased, perhaps due to sperm senescence. Pooling the sperm of several males partially compensated for the loss of fertilizing ability seen at the end of the reproductive period.

Animals↗
PubMed2001

Biological freezing of human articular chondrocytes.

AIM: To preserve viable, metabolically active chondrocytes cultured in alginate beads at -196 degrees C for further use in in vitro and in vivo studies. METHODS: Human articular chondrocytes were isolated from femoral condyles within 24 h post mortem. To optimize the biological freezing procedure, the chondrocytes were control-rate frozen in different concentrations of dimethyl sulfoxide (DMSO) in Dulbecco's MEM supplemented with 10% FCS before being thawed and the cell viability was determined by Trypan Blue exclusion test. To investigate the effect of control-rate freezing on chondrocyte metabolism, control-rate frozen chondrocytes in 5% DMSO were thawed and cultured in gelled agarose for 2 weeks. Non-frozen chondrocytes cultured in agarose served as controls. Furthermore, human articular chondrocytes were cultured in 2% alginate beads for 2 weeks after which the beads were incubated with 5% DMSO for 0 h, 2.5 h, 5 h and 10 h and frozen at -196 degrees C. Non-frozen alginate beads containing chondrocytes and incubated with 5% DMSO served as a control. After 2 weeks in culture, chondrocytes in agarose or in alginate were sulfated with 10 microCi(35)SO(4)/ml for 48 h. The total production of aggrecans, and the aggrecan subtypes, were subsequently determined. RESULTS: Five percent DMSO in the culture medium was the optimal condition to control-rate freeze and recover viable and functional isolated chondrocytes. Total aggrecan synthesis of control-rate frozen chondrocytes cultured in gelled agarose was not significantly reduced when compared with control cells. The proportion of aggrecan in the aggregate form of control-rate frozen chondrocytes kept in agarose remained unaltered. Chondrocytes, control-rate frozen in the alginate matrix, showed a 0-30% decrease in total aggrecan synthesis rates in culture when compared with the non-frozen chondrocytes. The optimal pre-incubation time of the alginate beads with 5% DMSO was 5 h, without any change in aggrecan synthesis rates when compared with the control situation. Shorter pre-incubation times resulted in an insufficient diffusion of DMSO into the beads and in cell death. There was no difference in the synthesis of the different aggrecan subtypes between frozen and non-frozen chondrocytes in alginate. CONCLUSION: Human articular chondrocytes can be stored at -196 degrees C for 24 h without important decreases in their aggrecan synthesis rates when control-rate frozen as a cell suspension in 5% DMSO. Proportions of the aggrecan subtypes (monomers, aggregates) synthesized by chondrocytes cultured in agarose remained unchanged. The control-rate freezing procedure in the alginate beads pre-incubated with 5% DMSO for 5 h produced no decrease in total aggrecan synthesis rates and no change in the synthesized aggrecan subtypes. Further experiments have to confirm the suitability of this freezing method for long-term storage of chondrocytes allowing us to set up a 'chondrocyte' bank for further use in in vitro and in vivo manipulations.

Adult↗
PubMed1990

Examination of methods for the deep freezing of goat semen.

In four experiments, freezing of goat semen in straws was examined and compared with conventional pellet-freezing. The optimum freezing regime was attained by holding straws in vapour 4 cm above liquid nitrogen for 30 s or longer, followed by plunging into liquid nitrogen. Straws exposed to vapour for only 10 s did not cool sufficiently before plunging, and cell viability was seriously impaired. Initiation of ice crystallization and the pattern of cooling of semen depended on size of straws and the type of rack used to hold straws in vapour during freezing. Nevertheless, semen was equally well stored in straws of 0.25 mL and 0.50 mL capacity and straws were most efficiently filled at 30 degrees C. Modified Tris media were used to dilute semen at rates (semen: diluent) of 1:2, 1:5, 1:11 and 1:23. Spermatozoa survived best in hypertonic diluents and deteriorated during post-thawing incubation in media of low tonicity, whilst the optimum diluent depended on rate of extension before freezing. The cooling curves differed markedly for semen frozen in straws in liquid nitrogen vapour and pellets on dry ice. Viability of spermatozoa was better for the pellet-frozen semen.

Animals↗
PubMed1993

The unique stability of the marsupial sperm acrosomal membranes examined by unprotected freeze-thawing and treatment with the detergent Triton X-100.

In this study of the unique stability of the marsupial acrosome, experiments were carried out on the acrosomes of spermatozoa of the tammar wallaby (Macropus eugenii), common brushtail possum (Trichosurus vulpecula) and grey short-tailed opossum (Monodelphis domestica). Light microscopy showed that 4% of opossum and 15% of possum and wallaby spermatozoa lost their acrosomes after freeze-thawing. Electron microscopy revealed that freeze-thawing also induced changes in the acrosomal matrix of some acrosome intact spermatozoa. In both possum and wallaby, freeze-thawing increased the number of spermatozoa with vesiculation of the acrosomal matrix. Freeze-thawing disrupted the plasma membrane of spermatozoa but the acrosomal membranes remained intact. Immediately on addition of high concentrations of TX-100 (0.02% and 0.04%) there was significant loss of acrosomes and motility in possum and wallaby spermatozoa. Lower concentrations of TX-100 (< or = 0.01%) did not affect motility for up to 30 min in all three species, and there was no significant loss of acrosomes. Although loss of acrosomes did not occur under mild detergent treatment, 56% of wallaby and 70% of possum spermatozoa had altered acrosomes after 30 min in 0.01% TX-100. Electron microscopy revealed that acrosomes were undergoing a vesiculation process similar to that seen after freeze-thawing. Often the plasma membrane of detergent-treated spermatozoa was disrupted and had formed plasma membrane vesicles. However, the acrosomal membranes remained intact despite major changes to the acrosomal matrix. The study confirmed the remarkable stability of the marsupial acrosome and suggested that this is probably based in the acrosomal membranes.

Acrosome↗
PubMed2004

Expression of an active tobacco mitogen-activated protein kinase kinase kinase enhances freezing tolerance in transgenic maize.

Cold acclimation is the major process that prepares plants for freezing tolerance. In addition to extensive transcription regulation by cold-inducible master transcription factors, oxidative stress signaling has been postulated to play a role in freezing tolerance. Activation of oxidative signaling through the expression of an active mitogen-activated protein kinase kinase kinase provided benefits in transgenic tobacco at freezing temperature bypassing cold acclimation. Because involvement of the mitogen-activated protein kinase cascade in oxidative stress signaling is evolutionarily conserved in eukaryotes from yeast to mammals, we tested the effect of expressing a heterologous tobacco mitogen-activated protein kinase kinase kinase (Nicotiana PK1), which can mimic H(2)O(2) signaling, in a major cereal crop. We demonstrate that low-level but constitutive expression of the Nicotiana PK1 gene enhances freezing tolerance in transgenic maize plants that are normally frost sensitive. Our results suggest that a new molecular approach can be designed to genetically enhance freezing tolerance in important crops.

Acclimatization↗
PubMed1977

Electron microscopy of negatively stained and freeze-etched high density lipoprotein-3 from human serum.

High density lipoproteins of d = 1.12 to 1.21 g/ml from human serum (HDL3) were studied by electron microscopy with both negative staining and freeze-etching techniques. For the negatively stained specimens, a modified conventional transmission electron microscope as well as a scanning transmission electron microscope were used. The freeze-etched specimens were examined by a conventional transmission electron microscope. The diameter of HDL3 was found to be 105 +/- 4 A by freeze-etching and 94 +/- 6 A by negative staining. The surface of the HDL3 particles exhibited about 12 discrete domains, 28 +/- 3 A (freeze-etched) and 28 +/- 4 A (negatively stained) in diameter, of undefined chemical composition. Moreover, the freeze-etched specimens revealed an inner core 40 +/- 2 A in diameter, corresponding to estimated values reported previously. All information is consistent with the HDL3 model proposed by B. W. Shen, F. J. Kézdy, and A. M. Scanu [(1977) Proc. Natl. Acad. Sci. USA 74, 837-841], with additional evidence for well-defined surface substructure. The consistency of the images obtained with the various electron microscopy techniques and the marked change in the appearance of the surface in the HDL3 preparations that were digested by phospholipase A2 (EC 3.1.1.4) support the validity of the interpretation.

Freeze Etching↗
PubMed2003

Cold-acclimation protects photosystem II against freezing damage in the halotolerant alga Dunaliella salina.

Cold-acclimation (CA) of the halotolerant alga Dunaliella was inhibited by light and by high salt. CA was associated with enhanced resistance to freezing in saline growth solutions, as manifested by protection of photosynthetic oxygen evolution and by reduced permeabilisation of the plasma membrane. Oxygen evolution activity in isolated chloroplasts was not affected by freezing, but was inhibited by high salt and the inhibition could be reversed or protected by glycerol. The activity of chloroplasts from cold-acclimated cells was more resistant to salt than of non-acclimated cells. Electron transport measurements in chloroplasts indicated that high salt inhibited PS-II, but not PS-I electron transport. High salt also inhibited PS-II thermoluminescence (TL) activity in chloroplasts. Similar inhibition of PS-II TL was observed by freezing intact cells in saline solutions. Chloroplasts from cold-acclimated cells had enhanced resistance to inhibition of PS-II electron transport and of PS-II TL by high salt. These results suggest that inhibition of oxygen evolution upon freezing Dunaliella cells may result from inactivation of PS-II due to massive influx of salt and loss of glycerol. The enhanced freeze-resistance of cold-acclimated cells to inhibition of oxygen evolution can be accounted for partly by protection of PS-II against high salt.

Acclimatization↗
PubMed2002

Determination of residues of oxolinic acid and flumequine in freeze-dried salmon muscle and skin by HPLC with fluorescence detection.

A procedure for the determination of residues of oxolinic acid (OA) and flumequine (FLU) in freeze-dried salmon muscle with attached skin, using reversed-phase high-performance liquid chromatography, is described. OA and FLU were extracted by a solid-liquid extraction procedure: after addition of hydrochloric acid, extraction used successively ethyl acetate, sodium hydroxide and chloroform. Liquid chromatography was performed on a 5 microm PuroSpher RP-18E cartridge using acetonitrile and 0.02 M aqueous orthophosphoric acid solution as mobile phase, with fluorescence detection. The performance of the method was established by spiking tissues with OA and FLU before the freeze-drying step. The method was linear over the concentration range 50-2000 ng/g freeze-dried tissue. Limits of detection and quantitation were 3.2 and 16 ng/g wet weight tissue respectively both for OA and FLU. Mean extraction recoveries of OA and FLU from freeze-dried tissue were 85.5 and 85.2% respectively. The method is suitable as a regulatory one for determination of residues of OA and FLU in freeze-dried salmon tissue.

Animals↗
PubMed1995

Stability of freeze dried horseradish peroxidase conjugated monoclonal antibodies used in diagnostic serology.

Two murine monoclonal antibodies, an IgG1 isotype specific for the heavy chain of bovine IgG1, the second an IgG3 isotype and specific for an epitope of the O-polysaccharide of Brucella abortus were conjugated with horseradish peroxidase. The conjugated antibodies were freeze dried in the presence of a number of additives to preserve activity and tested for stability over an 18 month period. Addition of 0.3M trehalose or 0.8% lactalbumin and 3.2% sucrose resulted in the lowest loss of activity if the conjugated antibodies were freeze dried in glass vials. Freeze drying in polypropylene vials resulted in a more rapid rate of deterioration with some additives or an accelerated rate of decline after an initial plateau of lesser loss of activity. The use of polypropylene vials and lactalbumin and sucrose were included in this study because of their low cost compared to glass vials and trehalose. While each antibody behaved differently, addition of trehalose or lactalbumin and sucrose to the conjugated antibodies aided in the preservation of enzyme activity. Both additives provide a suitable method for long term storage of freeze dried or freeze dried and reconstituted monoclonal antibody-enzyme conjugates.

Animals↗
PubMed2005

Hydroxyzine- and cetirizine-loaded liposomes: effect of duration of thin film hydration, freeze-thawing, and changing buffer pH on encapsulation and stability.

PURPOSE: To assess the effect of the duration of film hydration, freeze-thawing, and changing buffer pH on the extent of entrapment of hydroxyzine and cetirizine, H1-antihistamines with different polarity, into liposomes, and the stability of these liposomes. METHODS: Multilamellar vesicles (MLV) were prepared by thin-lipid film hydration using L-alpha-phosphatidylcholine (PC) and buffer containing 80 mg hydroxyzine at pH 7. For MLV containing hydroxyzine, the liposomes were subjected to 1) hydration for 1 h, 24 h, or 48 h for the control batch, batch B, or batch D respectively; and 2) hydration for 1 h, 24 h, or 48 h with freeze-thawing for 5-cycles for batch A, batch C, or batch E, respectively. These formulations were stored at 10 +/- 2 degrees C and 37 +/- 0.1 degrees C. Small unilamellar vesicles (SUV) and MLV were prepared using L-alpha-phosphatidylcholine (PC), and buffer at pH 5.0, 5.5, 6.0, 6.5, and 7.0, containing 80 mg hydroxyzine or 82 mg cetirizine by the ethanol injection and thin-lipid film hydration methods, respectively. These formulations were stored at 10 +/- 2 degrees C. Liposomes were evaluated immediately after preparation and after storage by determining percent entrapment of hydroxyzine (PETH) or of cetirizine (PEC) and by observing changes in the physical appearance (PA). Particle size (PSA) of the liposomes freshly prepared at pH=6.5 was measured from transmission electron micrographs (TEM). RESULTS: Increasing thin-film hydration time or repeated freeze-thawing did not affect the initial PETH or long-term stability of control, A, B, C, D, and E batches of MLV containing hydroxyzine stored at 10 +/- 2 degrees C. At 37 +/- 0.1 degrees C, PETH of all MLV batches decreased considerably after 1 month. This was more evident in batches B, C, and E exposed to freeze-thawing. The PETH of SUV increased markedly from 53.0% to 84.0% when the pH of the buffer was increased from 5.0 to 5.5. As pH increased from 6.0 to 7.0, PETH continued to increase from 84% to 94%. The initial PETH of MLV increased slightly from 82.0% to 94.0% as the buffer pH values increased from 5.0 to 7.0. There was no effect of pH on initial PEC, and stability of SUV or initial PEC of MLV, which ranged from 92% to 94%, as buffer pH values increased from 5.0 to 6.5. After storage at 10 +/- 2 degrees C PEC in MLV decreased from 94% to 74%. CONCLUSIONS: The freeze-thawing processes had some effect on the stability of liposomes stored at temperatures higher than ambient temperature, 37 +/- 0.1 degrees C. The effect of changing the buffer pH from 5.5 to 7.0, and from 5.0 to 6.5 on initial PETH and PEC, respectively, was minimal. After 24 months at l0 +/- 2 degrees C, pH had no effects on PETH; however, PEC of MLV decreased.

Cetirizine↗
PubMed1981

A 45Ca autoradiographic and stereological study of freeze-dried smooth muscle of the guinea pig vas deferens.

In an effort to more clearly elucidate the role of cellular structures as calcium sinks and sources in smooth muscle cells, the intracellular distribution of radioactive calcium was evaluated by a new method based on freeze-drying. The guinea pig vas deferens was exposed to a physiological salt solution that contained 45Ca. The muscle was then freeze-dried and prepared for electron microscope autoradiography. The grain density over the plasma membrane, mitochondria, and sarcoplasmic reticulum (SR) was significantly greater than that of the matrix. These results suggest that the plasma membrane, mitochondria and SR have the capacity to accumulate calcium. Which of these structures serve as a source of calcium for contraction remains to be determined. A stereological comparison between freeze-dried and conventionally prepared smooth muscles revealed several differences. The cross-sectional area of freeze-dried cells was about twice that of conventionally prepared cells. Moreover, mitochondria and sub-surface vesicles occupied a significantly smaller percentage of the cell in the freeze-dried tissue than they did in the conventionally prepared tissue.

Animals↗
PubMed1954

The effects on biological materials of freezing and drying by vacuum sublimation. II. Effect on influenza virus.

The infectivity titre of influenza virus-infected allantoic fluid was determined after a variety of procedures involving cyclic slow freezing and thawing, freezing at various rates with subsequent storage at different temperatures freezing at various rates with subsequent dehydration at various temperatures, and different degrees of dehydration. All these factors were found to influence the survival rate of the virus particles. Five freeze-thaw cycles resulted in a fall in titre from 10(-8.6) to 10(-0.8) cycles 2, 3, and 4 causing much greater losses than cycles 1 and 5. Rapid cooling to -40 degrees C. or slow cooling to -80 or 190 degrees C. did not cause significant titre loss, but rapid cooling to temperatures above -40 degrees or slow cooling to temperatures above -80 degrees C. caused definite titre loss. Loss of titre on storage occurred only at temperatures above -40deg;C. The effect of lyophilization depends both on the preliminary treatment and on the dehydration temperature. Better conservation of titre was obtained after preliminary cooling to -190 or -80 degrees C. than after preliminary cooling to higher temperatures. The most effective sublimation temperatures were 0 and -80 degrees .; the least effective was +20 degrees C. Titre losses in suspensions sublimated at -10, -30, and -60 degrees C. were in general intermediate. No loss in titre occurred after preliminary cooling to -80 or -190 degrees C. and subsequent dehydration at -80 or 0 degrees C. The degree of dehydration definitely affects the survival of virus on storage at 0 degrees C., but sublimation for 4 hours at 0 degrees C. gave complete protection against titre loss on storage at this temperature. Possible explanations of the observations made are suggested, based on known physiochemical phenomena such as supercooling, vitrification, variations in size and shape of ice crystals with different freezing speeds, differential enzyme inactivation, changes in salt concentration, and changes in energy levels.

Animals↗
PubMedDate not supplied

Freeze resistance in rainbow smelt (Osmerus mordax): seasonal pattern of glycerol and antifreeze protein levels and liver enzyme activity associated with glycerol production.

Rainbow smelt (Osmerus mordax) inhabit inshore waters along the North American Atlantic coast. During the winter, these waters are frequently ice covered and can reach temperatures as low as -1.9 degrees C. To prevent freezing, smelt accumulate high levels of glycerol, which lower the freezing point via colligative means, and antifreeze proteins (AFP). The up-regulation of the antifreeze response (both glycerol and AFP) occurs in early fall, when water temperatures are 5 degrees -6 degrees C. The accumulation of glycerol appears to be the main mechanism of freeze resistance in smelt because it contributes more to the lowering of the body's freezing point than the activity of the AFP (0.5 degrees C vs. 0.25 degrees C for glycerol and AFP, respectively) at a water temperature of -1.5 degrees C. Moreover, AFP in smelt appears to be a safeguard mechanism to prevent freezing when glycerol levels are low. Significant increases in activities of the liver enzymes glycerol 3-phosphate dehydrogenase (GPDH), alanine aminotransferase (AlaAT), and phosphoenolpyruvate carboxykinase (PEPCK) during the initiation of glycerol production and significant correlations between enzyme activities and plasma glycerol levels suggest that these enzymes are closely associated with the synthesis and maintenance of elevated glycerol levels for use as an antifreeze. These findings add further support to the concept that carbon for glycerol is derived from amino acids.

Alanine Transaminase↗
PubMed2006

Effect of freeze time during renal cryoablation: a swine model.

BACKGROUND AND PURPOSE: Cryotherapy provides a minimally invasive treatment for small renal tumors via an open, percutaneous, or laparoscopic approach. We sought to determine the most appropriate duration of freezing and the number of probes necessary to produce cell death without concomitant morbidity. MATERIALS AND METHODS: Nine domestic female pigs were divided into three groups of three animals each. Each group underwent a single freeze cycle with a commercially available cryotherapy device with 3.4-mm probes: group 1 for 5 minutes, group 2 for 10 minutes, and group 3 for 15 minutes. The right kidney was treated with a single probe, the left with a double probe. Animals were permitted to survive for an average of 4.8 days (range 4-7 days), after which the kidneys were harvested. A single pathologist examined the kidneys for gross and histologic changes. Evidence of complications (fistula, bleeding, bowel injury) was documented at the time of necropsy. RESULTS: For group 1, the temperature obtained with a single probe 5, 10, 15, and 20 mm from the probe was -57 degrees C, 3 degrees C, 25 degrees C, and 33 degrees C, respectively; for group 2 -85 degrees C, -37 degrees C, -2 degrees C, and 25 degrees C; and for group 3 -10 degrees C, -45 degrees C, -20 degrees C, and 6 degrees C. For group 1, the temperature obtained with a double probe at 5, 10, 15, and 20 mm from each probe was -65 degrees C, 0 degrees C, 20 degrees C, and 30 degrees C, respectively; for group 2 -72 degrees C, -25 degrees C, 5 degrees C, 25 degrees C; and for Group 3 -82 degrees C, -30 degrees C, -12 degrees C, 13 degrees C. Complete necrosis was seen 5 mm from the cryoprobe within each group, but only in groups 2 and 3 did necrosis extend 10 mm or beyond the probes when utilizing either single or double probes. The maximum diameter of consistent necrosis was 35 to 40 mm in the animals in group 3 treated with a double probe. Bleeding and renal fracture were the two most common complications. CONCLUSIONS: A 5-minute freeze appears to be inadequate to cause tissue necrosis and is associated with excessive bleeding at the time of the procedure, whereas the 15-minute freeze produces consistent necrosis but is associated with renal fracture. In this animal model, the 10-minute freeze with the single or double probe configuration appears optimal to produce necrosis without complications.

Animals↗
PubMed2006

Identification of cyanidin glycosides as constituents of freeze-dried black raspberries which inhibit anti-benzo[a]pyrene-7,8-diol-9,10-epoxide induced NFkappaB and AP-1 activity.

Dietary freeze-dried black raspberries inhibit tumor induction by N-nitrosomethylbenzylamine in the rat esophagus, but the constituents responsible for this chemopreventive activity have not been identified. We fractionated freeze-dried black raspberries and used mouse epidermal JB6 Cl 41 cells stably transfected with either a nuclear factor kappa B (NFkappaB)- or an activator protein 1 (AP-1)-luciferase reporter, and treated with racemic anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), to assess the inhibitory effects of the fractions. The ethanol and water extracts of the freeze-dried black raspberries had inhibitory activity and these extracts were fractionated by HPLC to give several bioactive fractions. Further HPLC analysis yielded multiple subfractions, some of which inhibited BPDE-induced NFkappaB activity. Major constituents of the most active subfractions were identified by their spectral properties and in comparison with standards as cyanidin-3-O-glucoside, cyanidin 3-O-(2(G)-xylosylrutinoside) and cyanidin 3-O-rutinoside. Analysis of freeze-dried black raspberries indicated that these three components comprised approximately 3.4% of the material by dry weight. Consistent with these results, standard cyanidin-3-O-glucoside and cyanidin chloride were also good inhibitors of BPDE-induced NFkappaB activity. The results of this study demonstrate that cyanidin glycosides of freeze-dried black raspberries are bioactive compounds which could account for at least some of the chemopreventive activity observed in animal models.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗