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F W Kleinhans

Publications and source records attributed to F W Kleinhans.

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

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. Glycerol was used as a model cryoprotective agent in the present study. To test this hypothesis, first the tolerance limits of spermatozoa to swelling in hypo-osmotic solutions (iso-osmotic medium diluted with water) and to shrinkage in hyperosmotic solutions (iso-osmotic medium with sucrose) were determined. Sperm plasma membrane integrity was measured by fluorescent staining, and sperm motility was assessed by computer-assisted semen analysis before, during and after the anisosomotic exposure. The result indicate firstly that motility was much more sensitive to anisosmotic conditions than membrane integrity, and secondly that motility was substantially more sensitive to hypotonic than to hypertonic conditions. Based on the experimental data, osmotic injury as a function of sperm volume excursion (swelling or shrinking) was determined. The second step, using these sperm volume excursion limits and previously measured glycerol and water permeability coefficients of human spermatozoa, was to predict, by computer simulation, the cell osmotic injury caused by different procedures for the addition and removal of glycerol. The predicted sperm injury was confirmed by experiment. Based on this study, an analytical methodology has been developed for predicting optimal protocols to reduce osmotic injury associated with the addition and removal of hypertonic concentrations of glycerol in human spermatozoa.

Buffers↗
PubMed1995

Effect of cryoprotectant solutes on water permeability of human spermatozoa.

Osmotic permeability characteristics and the effects of cryoprotectants are important determinants of recovery and function of spermatozoa after cryopreservation. The primary purpose of this study was to determine the osmotic permeability parameters of human spermatozoa in the presence of cryoprotectants. A series of experiments was done to: 1) validate the use of an electronic particle counter for determining both static and kinetic changes in sperm cell volume; 2) determine the permeability of the cells to various cryoprotectants; and 3) test the hypothesis that human sperm water permeability is affected by the presence of cryoprotectant solutes. The isosmotic volume of human sperm was 28.2 +/- 0.2 microns3 (mean +/- SEM), 29.0 +/- 0.3 microns3, and 28.2 +/- 0.4 microns3 at 22, 11, and 0 degrees C, respectively, measured at 285 mOsm/kg via an electronic particle counter. The osmotically inactive fraction of human sperm was determined from Boyle van't Hoff (BVH) plots of samples exposed to four different osmolalities (900, 600, 285, and 145 mOsm/kg). Over this range, cells behaved as linear osmometers with osmotically inactive cell percentages at 22, 11, and 0 degrees C of 50 +/- 1%, 41 +/- 2%, and 52 +/- 3%, respectively. Permeability of human sperm to water was determined from the kinetics of volume change in a hyposmotic solution (145 mOsm/kg) at the three experimental temperatures. The hydraulic conductivity (Lp) was 1.84 +/- 0.06 microns.min-1.atm-1, 1.45 +/- 0.04 microns.min-1.atm-1, and 1.14 +/- 0.07 microns.min-1.atm-1 at 22, 11, and 0 degrees C, respectively, yielding an Arrhenius activation energy (Ea) of 3.48 kcal/mol. These biophysical characteristics of human spermatozoa are consistent with findings in previous reports, validating the use of an electronic particle counter for determining osmotic permeability parameters of human sperm. This validated system was then used to investigate the permeability of human sperm to four different cryoprotectant solutes, i.e., glycerol (Gly), dimethylsulfoxide (DMSO), propylene glycol (PG), and ethylene glycol (EG), and their effects on water permeability. A preloaded, osmotically equilibrated cell suspension was returned to an isosmotic medium while cell volume was measured over time. A Kedem-Katchalsky model was used to determine the permeability of the cells to each solute and the resulting water permeability. The permeabilities of human sperm at 22 degrees C to Gly, DMSO, PG, and EG were 2.07 +/- 0.13 x 10(-3) cm/min, 0.80 +/- 0.02 x 10(-3) cm/min, 2.3 +/- 0.1 x 10(-3) cm/min, and 7.94 +/- 0.67 x 10(-3) cm/min, respectively. The resulting Lp values at 22 degrees C were reduced to 0.77 +/- 0.08 micron.min-1.atm-1, 0.84 +/- 0.07 micron.min-1.atm-1, 1.23 +/- 0.09 microns.min-1.atm-1, and 0.74 +/- 0.06 micron.min-1.atm-1, respectively. These data support the hypothesis that low-molecular-weight, nonionic cryoprotectant solutes affect (decrease) human sperm water permeability.

Cell Membrane Permeability↗
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

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

Determination of boar spermatozoa water volume and osmotic response.

Boar spermatozoa water volume and osmotic response were determined by a shapeindependent method for measuring cellular volume, electron paramagnetic resonance (EPR), employing the spin label, tempone, and the broadening agent, potassium chromium oxalate (CrOx). A water volume of 18.4 +/- 1.6 mum(3) (X +/- SD) was obtained for individual boar spermatozoa at 290 milliosmolar (mOsm) which, after correction for the presence of cytoplasmic droplets, yields a boar sperm water volume of 13.0 to 15.0 mum(3). Assuming 59% of the total cell volume is water, the total cell volume of boar spermatozoa is 22.0 to 25.4 mum(3). In addition, the experiment indicated that the relative water volume versus the reciprocal of the external osmolality (Boyle van't Hoff plot) was linear over the range of 210 to 1500 mOsm of sodium chloride (r(2) = 0.996), supporting the hypothesis that boar spermatozoa act as ideal osmometers. A non-zero y axis intercept of 0.23 from the Boyle van't Hoff plot indicated a 23% spin label accessible, but osmotically inactive water component.

Journal Article↗
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

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↗
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↗
PubMed1988

Electron spin resonance spectroscopy does not reveal hydroxyl radical production in activated natural killer lymphocytes.

We investigated hydroxyl radical (OH) production by human natural killer (NK) cells, using electron spin resonance (ESR) spectroscopy and 5.5 dimethyl-1-pyrroline-N-oxide (DMPO), a spin trap specific for OH. production. We confirmed that hydroxyl radical scavengers, n-propyl gallate and catechin, inhibited NK cell-mediated cytotoxicity (NK-CMC) in a dose-dependent manner and demonstrated that DMPO also inhibited NK-CMC. Polymorphonuclear leukocytes (PMNL) activated by opsonized zymosan (2.4 mg/ml) and mixed with DMPO (0.14 M) showed an early increase in hydroxyl radical production, leading to a net production of free radical of almost 400 pMol/10(6) cells. We then mixed NK cells with K562, an NK-sensitive tumor cell, at a 1:1 ratio and added DMPO (0.14 M). We pelleted the cells to increase EC to TC binding before taking the sample readings. Activated NK cells showed no increase in OH. production, leading to a net production of free radicals less than 1% that of activated PMNL. These data strongly suggest that hydroxyl radical production does not play a role in the early events of NK cell activation; they indicate a need to reevaluate the mechanism of inhibition of NK-CMC by OH. scavengers.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1987

Spin trap determination of free radical burst kinetics in stimulated neutrophils.

Electron spin resonance spin-trapping methods were used to investigate the free radical production kinetics of neutrophils stimulated with phorbol myristate acetate (PMA) and opsonized zymosan (OPZ). Using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide, the principle spin adduct observed is DMPO-OH (trapped hydroxyl radical). The DMPO-OH ESR signal amplitude was observed to decay exponentially. In such cases a simple method may be used to analyze the raw kinetics amplitude data to yield true production rate and net production data. The method, pitfalls, and self-consistency criteria are illustrated with PMA and OPZ-stimulated neutrophils at 25 and 37 degrees C under varying oxygen tensions, and with noise-free simulated data. The simulations demonstrate that rate results are relatively insensitive to the precise choice of decay time constant, tc, while net production results are very sensitive to the choice of tc used to analyze the raw data. OPZ (0.6-2.4 mg/ml) yields a strong, sharp neutrophil burst which peaks in 2 min or less while PMA yields a slower burst which peaks in 3.4-14 min for PMA concentrations of 500-50 ng/ml, respectively. Increased oxygen tension during the PMA experiments increased the spin adduct lifetime. The methods presented are applicable to other cell systems or spin adducts which exhibit first order decay.

Algorithms↗
PubMed1984

Comparative responses of human polymorphonuclear leukocytes obtained by counterflow centrifugal elutriation and Ficoll-Hypaque density centrifugation. II. Membrane potential changes, membrane receptor analysis, membrane fluidity, and analysis of the effects of the preparative techniques.

Standard preparative techniques for human polymorphonuclear leukocytes involves the sequential exposure of the PMN to dextran, Ficoll-Hypaque (FH) gradient centrifugation, and hypotonic stress. Counterflow centrifugal elutriation (CCE) allows isolation of PMN from whole blood without exposure to these potentially toxic substances. We have previously reported that PMN isolated by CCE release more superoxide on stimulation than do PMN isolated by standard techniques (FH PMN). In this report, we extend these observations and show that CCE and FH PMN have similar binding kinetics for the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) and similar membrane fluidity. CCE PMN, however, were noted to have a more marked membrane depolarization on stimulation with FMLP than FH PMN. We show that exposure of purified CCE PMN to dextran increases their superoxide release on stimulation, as well as the available FMLP binding sites. Exposure of FH PMN to elutriation buffer increased their superoxide release on stimulation. It was also noted that both CCE and FH PMN re-exposed to FH increased available binding sites for FMLP. The possible reasons for these findings are discussed.

Adult↗
PubMed1983

Determination of alternate pathway complement kinetics by electron spin resonance spectroscopy.

We describe a technic that measures the kinetics of the metabolic burst of peripheral blood neutrophils (PMN) by electron spin resonance (ESR) spin trapping. Using this technic, a functional assay for the kinetics of the alternate pathway complement cascade is developed. PMN were stimulated by phagocytosis of opsonized zymosan (OpZym) to undergo the metabolic burst and the resulting free hydroxyl radicals generated during the burst were trapped using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) to form a stable spin adduct. Rapid sequential measurements of the spin adduct concentration were made using a computer-controlled ESR spectrometer to follow the time course of the metabolic burst. Differences in the results obtained with OpZym and with PMN incubated with unopsonized zymosan (Zym) and serum yields information about the kinetics of opsonization. The kinetics of the alternate complement pathway opsonization in sera from normal and C8-deficient patients were compared and found not to be affected significantly by the absence of C8 complement protein.

Adult↗
PubMed1983

Comparison of electron paramagnetic resonance and atomic absorption serum copper measurements in human normal control and cancer patients.

Serum copper levels of 132 controls and 122 cancer patients (including Hodgkin's disease, lung cancer, breast cancer, leukemia, untreated patients, and patients in progression) were measured using both atomic absorption (AA) and electron paramagnetic resonance (EPR) techniques. The data pairs were compared using linear regression analysis, EPR versus AA, and all the data (controls and cancers) fit a single regression line with a squared correlation coefficient (r2) of 0.80. Comparison of the subpopulations revealed possible small differences, but none great enough to be of diagnostic value for individual patients. Thus, in a number of cases of practical interest, EPR-determined serum copper levels were essentially redundant with respect to AA measurements. Previous reports recommending the use and possible superiority of EPR for serum copper determinations appear not to have compared the EPR and AA techniques adequately or correctly. EPR serum copper measurements may yet provide unique data in these diseases, but a more detailed analysis of the spectral parameters will be required. EPR-determined serum transferrin levels are also reported.

Adult↗
PubMed1979

ESR determination of membrane order parameter in yeast sterol mutants.

ESR investigations designed to determine membrane order parameter in sterol mutants of Saccharomyces cerevisiae were conducted using the membrane probe, 5-doxyl stearic acid. These mutants are blocked in the ergosterol biosynthetic pathway and thus do not synthesize ergosterol, the end product sterol. They do not require exogenous ergosterol for growth and, therefore, incorporate ergosterol biosynthetic intermediates in their membrane. Increasing order parameter is reflective of an increase in membrane rigidity. Single mutants involving B-ring delta 8 leads to delta 7 isomerization (erg 2) and C-24 methylation (erg 6) showed greater membrane rigidity than wild-type during exponential growth. A double mutant containing both lesions (erg 6/2) showed an even greater degree of membrane rigidity. During stationary phase the order of decreasing membrane rigidity was erg 6 greater than erg 6/2 greater than erg 2 = wild-type. The increased membrane order parameter was attributed to the presence of substituted sterols rather than increased sterol content or altered fatty acid synthesis.

Cell Membrane↗
PubMed1979

Neisseria gonorrhoeae membrane microenvironment studied by spin-label electron spin resonance: comparison of colony types.

Spin-label electron spin resonance was used to characterize the microenvironment around spin probes which localize (i) in membranes, (ii) at the membrane surface, or (iii) in the cytoplasm of living Neisseria gonorrhoeae. Four colony types (T1, T2, T3, and T4) of gonococci were compared on the basis of the electron spin resonance parameters 2T parallel to, S (order parameter), and tau c (microviscosity). The concentration of spin label used had little or no effect on viability. T1 and T2 gonococci were found to have a more restricted environment for molecular motion of a membrane surface spin label than did T3 and T4. The membrane fluidity, as measured by a membrane lipid spin label, of T4 (S = 0.571) was significantly greater than that of T1 or T3 (S = 0.580). This difference was detected at 37 degrees C, at 25 degrees C, in agar-grown bacteria, and in exponential-phase cells. Studies using spin labels which probe different levels of the membrane indicated the presence of a membrane flexibility gradient. Cytoplasmic spin-label studies indicated that the cytoplasm of all gonococcal colony types was three to five times more viscous than water.

Cell Membrane↗
PubMed1976

The viscosity of mammalian nerve axoplasm measured by electron spin resonance.

1. The microviscosity of the axoplasm of can sciatic nerve was determined by an in vitro electron spin resonance (e.s.r.) method using the spin label tempone. To identify the spin label signal as one arising only from within the axoplasm, Ni2+ was used as a line broadening agent. In one series of experiments in nerves with sheath intact the Ni2+ ion was shown to eliminate the tempone signal arising from the surface water, and in another series of experiments, with the sheath slit, to eliminate the signal from the extracellular space as well. 2. A microviscosity of less than 5 centipoise (cP), i.e. 5x that of water, was determined for the axoplasm. Changes in the viscosity of the nerve axoplasm as a function of temperature over a range of 38 degrees down to 2 degrees C were seen to follow closely the viscosity change found for a water solution. 3. The microviscosity of nerve axoplasm and its change with temperature were related to axoplasmic transport of material in nerve fibres. The results were used to exclude a large increase in viscosity at low temperatures as the cause for the cold-block of fast axoplasmic transport.

Animals↗