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P Mazur

Publications and source records attributed to P Mazur.

At least 37 records · Page 2Linked to original sources
PubMed1995

The effect of collection temperature, cooling rate and warming rate on chilling injury and cryopreservation of mouse spermatozoa.

The experiments presented here identify several factors that affect survival (motility) of cryopreserved mouse spermatozoa after freezing and thawing. Among these factors are: (i) the temperature at which spermatozoa are collected, (ii) the cooling rate to 0 degrees C and (iii) the warming rate from -196 degrees C to ambient. When excised epididymides were cooled to near 0 degrees (1-4 degrees C) and spermatozoa collected and mixed with cryoprotectant at that temperature, motilities after subsequent freezing and thawing were 8-10 times higher than when the spermatozoa were collected from the epididymides at 22 degrees C. In addition, the survival rates of spermatozoa warmed at rates ranging from 150 to 2000 degrees C min-1 were about five times higher than those in suspensions warmed at about 7500 degrees C min-1. The combination of a low collection temperature and the lower warming rates resulted in approximately 50% motility relative to unfrozen controls. Motility was reduced to 6-8% when the collection temperature was 22 degrees C, and to approximately 10% when frozen suspensions of spermatozoa collected in the cold were rapidly warmed from -196 degrees C. When spermatozoa collected at 22 degrees C were abruptly cooled to 0 degrees C, 40-80% of the cells suffered an irreversible loss of motility after warming. In contrast, when spermatozoa were cooled to 0 degrees C at 1 degree C min-1 and warmed (either rapidly or slowly), motilities were similar to those of uncooled controls (75-90%).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

The Saccharomyces cerevisiae FKS1 (ETG1) gene encodes an integral membrane protein which is a subunit of 1,3-beta-D-glucan synthase.

In Saccharomyces cerevisiae, mutations in FKS1 confer hypersensitivity to the immunosuppressants FK506 and cyclosporin A, while mutations in ETG1 confer resistance to the cell-wall-active echinocandins (inhibitors of 1,3-beta-D-glucan synthase) and, in some cases, concomitant hypersensitivity to the chitin synthase inhibitor nikkomycin Z. The FKS1 and ETG1 genes were cloned by complementation of these phenotypes and were found to be identical. Disruption of the gene results in (i) a pronounced slow-growth phenotype, (ii) hypersensitivity to FK506 and cyclosporin A, (iii) a slight increase in sensitivity to echinocandin, and (iv) a significant reduction in 1,3-beta-D-glucan synthase activity in vitro. The nucleotide sequence encodes a 215-kDa polypeptide predicted to be an integral membrane protein with 16 transmembrane helices, consistent with previous observations that the etg1-1 mutation results in echinocandin-resistant glucan synthase activity associated with the nonextractable membrane fraction of the enzyme. These results suggest that FKS1 encodes a subunit of 1,3-beta-D-glucan synthase. The residual activity present in the disruption mutant, the nonessential nature of the gene, and results of Southern blot hybridization analysis point to the existence of a glucan synthase isozyme.

Base Sequence↗
PubMed1994

Human spermatozoa glycerol permeability and activation energy determined by electron paramagnetic resonance.

The permeability of human spermatozoa to glycerol and its activation energy were determined using electron paramagnetic resonance (EPR) techniques. EPR was used to monitor the aqueous cell volume change vs. time during the glycerol permeation process using the aqueous spin label 15N-tempone and the membrane impermeable broadening agent potassium trioxalatochromiate (chromium oxalate). The permeation process was completed in tens of seconds, requiring the use of a stopped-flow methodology. The glycerol permeability coefficient (Pg) was determined by fitting a simple theoretical model to the experimental data. The permeabilities of human spermatozoa in 1 molar and 2 molar glycerol at 20 degrees C are (10.3 +/- 0.3).10(-4) cm/min (mean +/- S.D.) and (6.0 +/- 1.4).10(-4) cm/min, respectively. The permeabilities of human spermatozoa in 2 molar glycerol at 30, 20, 10, and 0 degrees C are (8.3 +/- 1.3).10(-4) cm/min, (6.0 +/- 1.4).10(-4) cm/min, (2.1 +/- 0.4).10(-4) cm/min, and (1.1 +/- 0.3).10(-4) cm/min, respectively. The activation energy (Ea) for glycerol permeation between 30 degrees C and 0 degrees C was found to be 11.6 kcal/mol.

Cell Membrane Permeability↗
PubMed1994

Cis,cis-muconate lactonizing enzyme from Trichosporon cutaneum: evidence for a novel class of cycloisomerases in eucaryotes.

The absolute stereochemical courses of cis,cis-muconate lactonizing enzyme (MLE;EC 5.5.1.1) from Trichosporon cutaneum (TcMLE) and chloromuconate cycloisomerase (MLE II; EC 5.5.1.7) from Pseudomonas sp B13 have been determined from 1H NMR measurements. Both cycloisomerases convert cis,cis-muconate to (4S)-muconolactone by a syn lactonization, the absolute stereochemical outcome of which is identical to that observed with MLE from Pseudomonas putida. The regiochemical courses of cyclization of 3-halo-cis,cis-muconates by TcMLE and MLE II have been characterized and shown to differ in a halogen substituent dependent manner, suggesting at least a different active site architecture of the two MLEs. Moreover, the regiochemical preferences of MLE II and TcMLE parallel results previously observed for the nonenzymatic lactonization of the 3-halomuconates at pH 1-6 and in concentrated HCl, respectively, in which alternate mechanisms of cyclization were proposed [Pieken, W. A., & Kozarich, J. W. (1990) J. Org. Chem. 55, 3029-3035]. Complementary DNA clones encoding TcMLE have been isolated from phenol induced T. cutaneum cDNA using the polymerase chain reaction. The deduced amino acid sequence does not exhibit any similarity to that of MLE from P. putida. It does however, exhibit moderate sequence similarity (21% residue identity, 14 gaps) with 3-carboxy-cis,cis-muconate lactonizing enzyme (CMLE; EC 5.5.1.5) from Neurospora crassa, which catalyzes a regiochemically analogous and stereochemically identical lactonization reaction with 3-carboxymuconate. The limited data available suggest that the fungal CMLE and yeast MLE are representative of a unique class of eucaryotic cycloisomerases which have evolved convergently with the bacterial MLEs.

Amino Acid Sequence↗
PubMed1994

3-Carboxy-cis,cis-muconate lactonizing enzyme from Neurospora crassa: an alternate cycloisomerase motif.

3-Carboxy-cis,cis-muconate lactonizing enzyme (CMLE; EC 5.5.1.5) from Neurospora crassa catalyzes the reversible gamma-lactonization of 3-carboxy-cis,cis-muconate by a syn-1,2 addition-elimination reaction. The stereochemical and regiochemical course of the reaction is (i) opposite that of CMLE from Pseudomonas putida (EC 5.5.1.2) and (ii) identical to that of cis,cis-muconate lactonizing enzyme (MLE; EC 5.5.1.1) from P. putida. In order to determine the mechanistic and evolutionary relationships between N. crassa CMLE and the procaryotic cycloisomerases, we have purified CMLE from N. crassa to homogeneity and determined its nucleotide sequence from a cDNA clone isolated from a p-hydroxybenzoate-induced N. crassa cDNA library. The deduced amino acid sequence predicts a protein of 41.2 kDa (365 residues) which does not exhibit sequence similarity with any of the bacterial cycloisomerases. The cDNA encoding N. crassa CMLE was expressed in Escherichia coli, and the purified recombinant protein exhibits physical and kinetic properties equivalent to those found for the isolated N. crassa enzyme. We also report that N. crassa CMLE possesses substantially reduced yet significant levels of MLE activity with cis,cis-muconate and, furthermore, does not appear to be dependent on divalent metals for activity. These data suggest that the N. crassa CMLE may represent a novel eucaryotic motif in the cycloisomerase enzyme family.

Amino Acid Sequence↗
PubMed1994

Water volume and osmotic behaviour of mouse spermatozoa determined by electron paramagnetic resonance.

Experiments were conducted to determine the water volume and osmotic behaviour of mouse spermatozoa using an electron paramagnetic resonance technique using the spin label tempone, and the broadening agent potassium chromium oxalate. After a swim-up procedure, an average water volume of 43.3 micron3 of individual spermatozoa was obtained at 290 mosmol. If a water compartment of 59% is assumed, the total volume of mouse spermatozoa is 73.4 micron3. A plot of the relative water volume of mouse spermatozoa versus the reciprocal of buffer osmolality (Boyle van't Hoff plot) is linear in the range 250-900 mosmol of sodium chloride solutions (r2 = -.96). The Boyle van't Hoff plot intercept indicates that 13% of the spin-label accessible isotonic water is osmotically inactive.

Animals↗
PubMed1993

Contributions of cooling and warming rate and developmental stage to the survival of Drosophila embryos cooled to -205 degrees C.

Because of their high susceptibility to chilling injury, permeabilized Drosophila embryos can not be cryobiologically preserved by slow freezing at rates low enough to prevent the formation of intraembryonic ice. Calculations indicated that to outrun the chilling injury they must be cooled and warmed rapidly at an estimated 20,000 degrees C/min or faster. Ordinarily, such cooling rates would inevitably produce lethal intracellular ice. To prevent this, embryos must contain and be surrounded by sufficiently high concentrations of glass-promoting solutes to induce vitrification on cooling and prevent devitrification on warming. Like Steponkus et al. (Nature 345, 170, 1990) we have used ethylene glycol as the solute and have exposed permeabilized 12-h embryos to it in two steps. (Permeabilization was effected by exposing dechorionated embryos to a mixture of 0.3% 1-butanol in n-heptane for 90 or 110 s.) The two steps were (i) a 30-min exposure to 2 M ethylene glycol at 23 degrees C and (ii) a 5-min exposure to 8.5 M ethylene glycol [+/- 10% polyvinylpyrrolidone (PVP)] at 5 degrees C. The volumetric response to the first step indicates that full permeation of the 2 M glycol has been approached by 30 min. The point of the second step is to raise the intraembryonic concentration of ethylene glycol to near 8.5 M ethylene glycol by osmotic dehydration. Survival based on hatching is some 45% at this point. When 12-h embryos in 8.5 M glycol containing 10% PVP are then cooled to -205 degrees C at approximately 100,000 degrees C/min and warmed at about that rate, an average of about 12% survive (hatch), although in about half the runs 15-29% survive. Survivals in the absence of PVP are usually poorer but have been as high as 40%. Currently, 5% of the surviving larvae develop to adult flies (Steponkus et al. reported 18% hatching and 3% development to adult). Embryos that develop but do not hatch show readily detectable abnormalities in mouth parts and dorsal closure. Very high warming rates are much more critical to survival than are very high cooling rates; for example, none survive when warming is 2000 degrees C/min. The deleterious effect of slow warming is exerted between -80 and -40 degrees C. The lack of reciprocity between the effects of time spent cooling and time spent warming argues against ascribing death to chilling injury. Rather, it and other data argue for ascribing death to the devitrification during warming of cytoplasm that vitrified during cooling.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Expression, purification, and characterization of recombinant ornatin E, a potent glycoprotein IIb-IIIa antagonist.

A synthetic gene encoding ornatin E (OrnE), a 50-amino acid glycoprotein IIb-IIIa (GP IIb-IIIa) antagonist and platelet aggregation inhibitor isolated from the leech Placobdella ornata, was designed, constructed, and expressed in Escherichia coli. The OrnE gene was fused to the heat stable enterotoxin stII signal sequence and expressed under the transcriptional control of the E. coli alkaline phosphatase promoter. This construction directed secretion of recombinant ornatin E (rOrnE) into the extracellular medium at levels of 7-19 mg/liter. The protein was purified to apparent homogeneity in 18-38% yields by ammonium sulfate precipitation, Q-Sepharose and S-Sepharose ion exchange chromatography, and reverse-phase HPLC. Purified rOrnE was found to be indistinguishable from leech-derived OrnE as judged by amino acid composition, N-terminal sequencing, mass spectroscopic analysis, and HPLC coelution. In addition, rOrnE exhibits similar activity in fibrinogen/GP IIb-IIIa ELISA and platelet aggregation assays. Purified rOrnE possesses three disulfide bonds, the reduction and carboxymethylation of which results in a ca. 60-fold reduction in biological activity. A misfolded variant of rOrnE was characterized and shown to have a ca. 6-fold reduction in activity. These data demonstrate that the native disulfide bonds are required for the optimal GP IIb-IIIa antagonist activity of the ornatins.

Amino Acid Sequence↗
PubMed1993

Invited commentary: cryopreservation as a tool for the study of selectively bred lines in rodent behavioral genetics.

Cryopreserved embryos should survive centuries. Upon rederivation, in vitro survival is high. Upon reimplantation, in vivo survival is substantial. Genetic stability of the embryos at -196 degrees C is theoretically absolute for several decades and likely high for centuries. Cryopreservation and rederivation services are available at a reasonable cost from commercial vendors. Preservation of a sufficient number of embryos can serve as an insurance policy against loss of the lines from several causes. In addition to the protective utility of a single banked sample, repeated preservation during the course of selection offers several advantages. Correlated responses to selection which appear late in selection may be verified by use of a cross-sectional experimental design to take a "snapshot" of the gradually developing response. After attainment of the selection limit, a generation may be chosen retrospectively which represents the best compromise between maximizing response and minimizing inbreeding irrelevant to the selected trait. Estimates of changes in gene frequencies could be derived from molecular biological analyses. An example of such an experiment in progress employing embryos from the COLD and HOT selected mouse lines is discussed.

Animals↗
PubMed1993

Determination of water permeability coefficient for human spermatozoa and its activation energy.

Four experiments were conducted to determine the permeability coefficient of human sperm to water (Lp) and its activation energy (Ea). Critical tonicity (tonicity at which 50% of the cells swell and lyse) was determined by equilibrating sperm to 22 degrees C (experiments 1a and 1b), 30, 22, 8, or 0 degrees C (experiment 2a), and 0, -1, -3, -5, or -7 degrees C (experiment 2b) and then exposing them to various hypotonic media (215-3 mOsm). For Lp determination, sperm were equilibrated to 30, 22, 8, or 0 degrees C (experiment 3a), 8, 0, or -3 degrees C (experiment 3b), and -1, -3, -5, or -7 degrees C (experiment 3c), and then were exposed for increasing times to hypotonic (40 mOsm) media. Activation energies were calculated from the results of the latter experiments (experiment 4). Results indicate a temperature-dependent (p < 0.05) critical tonicity, with sperm exhibiting an increased membrane fragility at 8, 0, and -7 degrees C, relative to 30, 22, -1, -3, or -5 degrees C (67.5 +/- 2.4, [mean +/- SEM], 62.7 +/- 2.3, and 61.9 +/- 3.7 mOsm vs. 57.4 +/- 3.4, 57 +/- 1.2, 54.8 +/- 3.4, 60.1 +/- 5.3, and 59.8 +/- 5.2 mOsm, respectively). Human sperm have an Lp of 2.40 +/- 0.20 microns/min/atm at 22 degrees C and an Ea of 3.92 +/- 0.59 kcal/mol between 30 and -7 degrees C. The Ea for cells incubated at temperatures above 0 degrees C (3.92 kcal/mol) show an apparent discontinuity (p < 0.004) in water permeability in supercooled conditions (7.48 kcal/mol). These data suggest that 1) human sperm have a high Lp and low Ea, relative to other cell types, above 0 degrees C; and 2) this high Lp and its low Ea change significantly below 0 degrees C.

Adult↗
PubMed1993

Hyperosmotic tolerance of human spermatozoa: separate effects of glycerol, sodium chloride, and sucrose on spermolysis.

Hyperosmotic stress, which cells experience during the freezing process, and its release during the warming process are both related to cryoinjury. To define optimal cooling or warming rates and prevent osmotic injury to human sperm, information is required regarding the osmotic tolerance of the cells as a function of 1) time, 2) temperature, 3) type of solute, and 4) solute concentration. Human sperm samples were divided into three aliquots. The aliquots were equilibrated at 0, 8, and 22 degrees C, respectively. Different hyperosmotic solutions were prepared by addition of either a permeating cryoprotective agent (glycerol) or nonpermeating solutes (sucrose, non-ionic; or NaCl, ionic) to isotonic Mann's Ringer solution. Aliquots of the prepared solutions were equilibrated at 0, 8, and 22 degrees C, respectively. A small volume (2.5 microliters) of each sperm aliquot was quickly mixed with 50 microliters of each hyperosmotic solution at the corresponding temperature. After times ranging from 5 s to 5 min, 10 microliters of each hyperosmotic cell suspension was abruptly returned to an isosmotic environment by mixing with 500 microliters of Mann's Ringer solution at the corresponding temperature. The plasma membrane integrity of cells after exposure to hyperosmotic stress and after return to isosmotic conditions was measured by a dual staining (carboxyfluoroscein diacetate and propidium iodide) technique and flow cytometry. The morphology of the treated cells was observed by scanning electron microscopy of freeze-substituted sperm. The results indicate that human spermatozoa exhibited a significant posthypertonic lysis/injury, i.e., loss of membrane integrity, when returned to isosmotic conditions after exposure to hyperosmotic solutions of NaCl or sucrose. The higher the hyperosmolality, the more serious the cell injury. The majority of the cells (> 50%) lost membrane integrity when the osmolality was > or = 2000 mOsm. In contrast, if the sperm were not returned to isosmotic conditions, the majority of the sperm in the hyperosmotic solutions appeared to maintain membrane integrity. For a given higher hyperosmolality (> 1000 mOsm), posthypertonic spermolysis was reduced with a decrease of temperature. Cell survival was also affected by time of cell exposure to hyperosmotic environments before cells were returned to the isotonic condition. The shorter the time, the higher the cell survival. When exposed to hyperosmotic glycerol solutions that were isotonic with respect to electrolytes, few cells lost their membrane integrity if the osmolality of glycerol was < 3000 mOsm. For a fixed high osmolality (> 3000 mOsm), the lower the temperature, the higher the percentage spermolysis.(ABSTRACT TRUNCATED AT 400 WORDS)

Body Water↗
PubMed1993

Cryopreservation of human spermatozoa. IV. The effects of cooling rate and warming rate on the maintenance of motility, plasma membrane integrity, and mitochondrial function.

OBJECTIVE: To test the hypotheses that there is a two-factor aspect of cellular damage during cryopreservation that occurs in human sperm (osmotic effects versus intracellular ice formation) and that there is a cooling rate by warming rate interaction related to this damage. DESIGN: Ejaculates from healthy men were cooled at 0.1, 1.0, 10, 175, or 800 degrees C/min to -80 degrees C in a solution of 0.85 M glycerol and plunged into liquid nitrogen. Samples were warmed at 400 degrees C/min (experiment 1) or either 1 degrees C or 400 degrees C/min (experiment 2). After warming, sperm were assessed for survival using motility as the endpoint in experiment 1 and motility, plasma membrane integrity, and mitochondrial function in experiment 2. RESULTS: In experiment 1, over the various cooling rates with a standard 400 degrees C/min warming rate, a plot of motility versus cooling rate produced a classical inverted U-shaped curve (n = 6) with maximum motility at the 10 degrees C/min cooling rate. In experiment 2, over the various cooling rates, both 1 and 400 degrees C/min warming rates produced similar but shifted plots of motility, plasma membrane integrity, and mitochondrial function versus cooling rate, which also produced inverted U-shaped patterns (n = 11). Maximal survival for each of the three endpoints occurred at 10 degrees C/min cooling rate for the rapidly warmed sperm and at 1 degree C/min for the slowly warmed sperm. CONCLUSIONS: These data support the hypotheses that a two-factor hypothesis of cryodamage applies to human spermatozoa and that an interaction exists between cooling rate and warming rate. These data also suggest that motility, plasma membrane integrity, and mitochondrial function are not differently affected by cooling and warming during cryopreservation.

Adult↗
PubMed1992

Cryobiological preservation of Drosophila embryos.

The inability to cryobiologically preserve the fruit fly Drosophila melanogaster has required that fly stocks be maintained by frequent transfer of adults. This method is costly in terms of time and can lead to loss of stocks. Traditional slow freezing methods do not succeed because the embryos are highly sensitive to chilling. With the procedures described here, 68 percent of precisely staged 15-hour Oregon R (wild-type) embryos hatch after vitrification at -205 degrees C, and 40 percent of the resulting larvae develop into normal adult flies. These embryos are among the most complex organisms successfully preserved by cryobiology.

Animals↗
PubMed1992

Characteristics and kinetics of subzero chilling injury in Drosophila embryos.

Drosophila embryos manifest unusually high sensitivity to chilling in that they are killed with increased rapidity by exposure to temperatures between 0 and -25 degrees C in the absence of ice formation. Thus, 50% of 15-h eggs succumb in 35, 4, and 1 h at 0, -9, and -15 degrees C, respectively. The sensitivity becomes substantially greater in embryos at stages of development earlier than 12 h, especially at 3 and 6 h. The killing kinetics at given subzero temperatures between 0 and -25 degrees C are characterized by a shoulder followed by a more-or-less linear decrease in survival with time. The lower the temperature, the shorter the shoulder and the faster the postshoulder decline. The rate of both components follows Arrhenius kinetics, i.e., plots of log rate vs 1/absolute temperature are linear, the slopes being proportional to the activation energy. In both cases the activation energy is high and negative; namely, -46.5 kcal/mol for the shoulder length and -24.7 kcal/mol for the postshoulder inactivation. Negative activation energies are unusual, and according to absolute reaction rate theory, they exist only when the entropy of activation is negative, which suggests that the activated state is more ordered. By combining the duration of the shoulder as a function of time and temperature with the rate of postshoulder inactivation, one can compute survival as a function of temperature for embryos cooled at various rates. For those cooled at less than or equal to 1 degree C/min, the computed curve of survival vs temperature agrees closely with observed survivals. But for embryos cooled at approximately 10 degrees C/min, the drop in survival occurs some 7 to 10 degrees above that computed. Embryos exposed to 0 degree C for greater than 5 min undergo conditioning that renders them more resistant to subsequent exposure to lower temperatures, and those cooled at 10 degrees C/min presumably lack sufficient time at 0 degree C to undergo such conditioning; hence the discrepancy between observed and computed survivals. As a test of the possibility that chilling injury is a consequence of the loss of synchrony of coupled reactions involved in embryological development, embryos were rendered anoxic prior to chilling, a treatment that has been shown by Foe and Alberts to reversibly halt development of early stages. Although anoxia somewhat reduced chilling injury in 6-h eggs, it had no effect on 15-h eggs.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Critical factors affecting the permeabilization of Drosophila embryos by alkanes.

Because of waxes in the vitelline membrane, the Drosophila egg is effectively impermeable to liquid water and to aqueous solutes, and consequently it cannot be cryopreserved unless it can be permeabilized. The more successful of the few published permeabilization procedures involve the removal of the chorion mechanically or by hypochlorite solution, the removal of all surrounding water by air drying or alcohol, the exposure of eggs to pure alkanes like octane or hexane for some 30 s, the removal of the alkane and the transfer of the eggs to aqueous culture medium without their desiccation, and lastly incubation of the permeabilized embryos under mineral oil. In following these procedures we opted for a somewhat different approach to applying hypochlorite, water, alcohol, and alkane; namely, eggs were placed between two Nucleopore filters, and the fluids drawn sequentially through the filters by vacuum. Extensive initial attempts were mystifying and discouraging in that although permeabilization was good, survivals were poor, and modifications that increased the latter reduced the former. The explanation turned out to be that permeabilization and survival depended critically on the amount of carry-over alcohol that contaminated the alkane. To determine the effects of alcohol concentration in the alkane, it was essential first to effectively eliminate carry-over contamination and then re-add precise amounts of alcohol (isopropanol) to the alkane (n-hexane, heptane, or octane). When the alcohol concentration is less than or equal to 0.2%, permeabilization is poor; when it is greater than or equal to 0.5%, permeabilization is good but survival (hatching) is poor. There are strong interactions between alcohol concentration and exposure time to alkane/alcohol mixtures with respect to the fraction of embryos that become permeabilized and the percentage that survive. There are also significant but less critical effects from the type of alcohol and alkane. The best results for 12-h embryos (greater than or equal to 90% permeabilization and 70-80% hatching) were achieved with eggs exposed to 0.3 or 0.4% 1-butanol in n-heptane for 90 s. High survivals of permeabilized 12-h embryos did not require incubation under mineral oil. Permeabilized embryos are permeable to water, ethylene glycol, glycerol, and the stain rhodamine B (which was used to assess permeabilization). They are effectively impermeable to sucrose. Embryo age is important. Between 14 and 16 h the above permeabilization procedures become dramatically less effective.(ABSTRACT TRUNCATED AT 400 WORDS)

Air↗
PubMed1992

Glycerol permeability of human spermatozoa and its activation energy.

Glycerol has commonly been employed as a cryoprotectant in cryopreservation of human spermatozoa. However, the addition of glycerol into the sperm before freezing and the removal of glycerol from the sperm after freezing and thawing result in anisotonic environments to the cells, which can cause cell injury. To define optimal procedures for the addition/removal of glycerol and to minimize the cell injury, one needs to know the kinetics of glycerol permeation across the sperm plasma membrane at different temperatures. For this, one has to determine the permeability coefficient of glycerol (Pg) and its activation energy (Ea). Values of Pg at different temperatures and at different glycerol concentrations were determined by measuring the time required for 50% spermolysis in hyperosmotic glycerol solutions which were hypotonic with respect to electrolytes. Value of the Ea was determined assuming an Arrhenius type temperature dependence of Pg. A dual fluorescent staining technique (propidium iodide and 6-carboxyfluoroscein diacetate) and flow cytometry were used to measure the spermolysis. The values of Pg in 0.5, 1.0, 1.5, and 2.0 M glycerol at 22 degrees C are 1.62, 1.88, 1.68, and 1.54 x 10(-3) cm/min, respectively. The values of Pg in 1 M glycerol at 0, 8, 22, and 30 degrees C are 0.33, 0.54, 1.88, and 2.60 x 10(-3) cm/min, respectively. The value of Ea is 11.76 kcal/mol.

Cell Death↗
PubMed1991

Ornatins: potent glycoprotein IIb-IIIa antagonists and platelet aggregation inhibitors from the leech Placobdella ornata.

The purification and characterization of six isoforms of ornatin, potent glycoprotein IIb-IIIa (GP IIb-IIIa) antagonists and platelet aggregation inhibitors are described. These isoforms were purified from whole leech homogenates of the leech Placobdella ornata, a North American leech commonly known as the turtle leech, by trichloroacetic acid precipitation, Sephadex G-50 size exclusion chromatography, GP IIb-IIIa affinity chromatography, and C18 reverse-phase HPLC. Each of the five completely sequenced isoforms, which range from 41 to 52 residues in length, contains the Arg-Gly-Asp (RGD) sequence, a common recognition sequence in adhesion proteins, as well as 6 cysteine residues; the positions of both of these features are conserved in the primary sequences. The amino acid sequences of ornatin isoforms B, C, D, and E are highly conserved, whereas ornatin A2 and A3 are less similar and lack 9 residues at the N-terminus. The ornatins are approximately 40% identical with decorsin, a GP IIb-IIIa antagonist isolated from the leech Macrobdella decora [Seymour, J. L., Henzel, W. J., Nevins, B., Stults, J. T. & Lazarus, R. A. (1990) J. Biol. Chem. 265, 10143-10147]; furthermore, the RGD sequence and 5 out of 6 cysteine residues are maintained in the same relative positions in both decorsin and ornatin. The ornatin isoforms do not exhibit significant similarity to any members of the snake-venom-derived family of GP IIb-IIIa antagonists [Dennis, M. S., Henzel, W. J., Pitti, R. M., Lipari, M. T., Napier, M. A., Deisher, T. A., Bunting, S. & Lazarus, R. A. (1990) Proc. Natl Acad. Sci. USA 87, 2471-2475] except in the RGD region of these proteins. The ornatin isoforms inhibit the binding of GP IIb-IIIa to immobilized fibrinogen with IC50 values ranging over 2.9-5.3 nM; ornatin isoforms A2, C, and E inhibit ADP-induced human platelet aggregation with IC50 values of about 130, 280, and 440 nM, respectively.

Amino Acid Sequence↗