Osmotic tolerance limits and properties of murine spermatozoa.

Osmotic tolerance of spermatozoa is a critical determinant of functional survival after cryopreservation. This study first tested the hypothesis that mouse spermatozoa behave as linear osmometers, using an electronic particle counter to measure the change in sperm volume in response to anisosmotic solutions. The resulting Boyle-van't Hoff plot was linear (r2 = 0.

In vitro activity of 1,3-beta-D-glucan synthase requires the GTP-binding protein Rho1.

In the yeast Saccharomyces cerevisiae, the family of RHO genes are implicated in the control of morphogenetic events although the molecular targets of these GTP-binding proteins remain largely unknown. The activity of 1,3-beta-D-glucan synthase, the product of which is essential for cell wall integrity, is regulated by a GTP-binding protein, which we here present evidence to be Rho1p. Rho1p was found to copurify with Fks1p, a glucan synthase subunit, in preparations of the enzyme purified by product entrapment and was also shown to be depleted by a detergent extraction procedure known to remove the GTP-binding regulatory component.

Permeabilization of eggs of the malaria mosquito Anopheles gambiae.

Although Anopheles gambiae embryos that have developed for 15 h at 17 degrees C are slightly permeable to water, they are impermeable to ethylene glycol, the cryoprotectant used in the cryopreservation of Drosophila melanogaster embryos. Success in cryopreservation requires that they be made permeable to protective solutes. Permeabilization of D.

Permeability of intact and dechorionated eggs of the Anopheles mosquito to water vapor and liquid water: a comparison with Drosophila.

As an initial step in the development of cryopreservation methods for Anopheles gambiae embryos, we determined the permeability of intact and dechorionated A. gambiae embryos to water and ethylene glycol and compared those permeabilities with those of Drosophila melanogaster embryos. Most studies were conducted on embryos allowed to develop for 8 h at 26 degrees C or 15 h at 17 degrees C.

Crystallization and preliminary crystallographic analysis of 3-carboxy-cis,cis-muconate lactonizing enzyme from Neurospora crassa.

Crystals of 3-carboxy-cis,cis-muconate lactonizing enzyme (CMLE; E.C. 5.

Spectral tuning in bacteriorhodopsin in the absence of counterion and coplanarization effects.

The basis for wavelength regulation in bacteriorhodopsin (BR) and retinylidene proteins in general has been studied for decades but is still only partially understood. Here we report the preparation and spectroscopic characterization of BR analogs aimed at investigating the existence of spectral tuning mechanisms other than the two widely accepted mechanisms, weakened counterion interactions and ring/chain coplanarization. We synthesized two novel retinal analogs containing a saturated 13-14 bond, which interrupts the interaction of the protein counterions with the chromophore conjugation system.

Differential expression and function of two homologous subunits of yeast 1,3-beta-D-glucan synthase.

1,3-beta-D-Glucan is a major structural polymer of yeast and fungal cell walls and is synthesized from UDP-glucose by the multisubunit enzyme 1,3-beta-D-glucan synthase. Previous work has shown that the FKS1 gene encodes a 215-kDa integral membrane protein (Fks1p) which mediates sensitivity to the echinocandin class of antifungal glucan synthase inhibitors and is a subunit of this enzyme. We have cloned and sequenced FKS2, a homolog of FKS1 encoding a 217-kDa integral membrane protein (Fks2p) which is 88% identical to Fks1p.

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.

Prevention of osmotic injury to human spermatozoa during addition and removal of glycerol.

Use of a cryoprotective agent is indispensable to prevent injury to human spermatozoa during the cryopreservation process. However, addition of cryoprotective agents to spermatozoa before cooling and their removal after warming may create severe osmotic stress for the cells, resulting in injury. The objective of this study was to test the hypothesis that the degree (or magnitude) of human sperm volume excursion can be used as an independent indicator to evaluate and predict possible osmotic injury to spermatozoa during the addition and removal of cryoprotective agents.

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.

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).

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.

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.

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.

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.

[The characteristics of medical supplies under current conditions].

The present situation requires that medical supply system could make it possible to assure an adequate ratio in distribution of the resources, using stable economic links between all the subjects on the basis of objective economic laws. As the world experience shows the best form for this matter is wholesale trade of medications and medical technique. Nevertheless, it is impossible to implement this form of supply without detailed and complex researches in the field of economics for military medicine and pharmacy, and reconstructing on this basis the system of training of commanding officers for military medical supply.

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.

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.

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.

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.

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.