Botulinum Neurotoxin Light Chain Inhibits Norepinephrine Secretion in PC12 Cells at an Intracellular Membranous or Cytoskeletal Site

Botulinum neurotoxin (NT) is a potent inhibitor of neurotransmitter secretion, but its intracellular mechanism and site of action are unknown. In this study, the intracellular action of NT was investigated by rendering the secretory apparatus of PC12 cells accessible to macromolecules by a recently described "cell cracking" procedure. Soluble cytoplasmic factors were depleted from permeabilized cells by washing to generate cell "ghosts" which retained cellular structural components and intracellular organelles (including secretory granules). The PC12 cell ghosts exhibited Ca(2+)-activated [3H]norepinephrine release which was enhanced by cytosolic proteins and MgATP. PC12 cell ghosts provide the opportunity to distinguish the intracellular action of NT on soluble cytoplasmic components versus structural cellular components. The 150-kDa NT and the 50-kDa light chain of serotypes E and B, and to a lesser extent type A, inhibited Ca(2+)-activated [3H]norepinephrine release in PC12 ghosts, but not in intact PC12 cells. The 100-kDa heavy chain had no effect. This indicates that NT acts at an intracellular site in these cells permeabilized by "cell cracking." The inhibition of secretion by NT was rapid and irreversible under the incubation conditions used. NT inhibition of [3H]-norepinephrine release from PC12 ghosts occurred in the absence of cytosolic proteins and MgATP and was not reversed by the addition of cytosolic proteins and MgATP, indicating that NT acts at an intracellular membranous or cytoskeletal site.     

Cell volume and osmotic properties of erythrocytes after complement lysis measured by flow cytometry

The changes of volume distribution curves of erythrocytes during and after lysis by complement or nystatin or in hypotonic buffers were measured by flow cytometry. Biconcave and spheroidal ghosts were observed after complement lysis and spheroidal ghosts were seen only after nystatin and hypotonic lysis. The spheroidal ghosts derived from red cells lysed by complement or nystatin were permeable to sucrose; those from hypotonic lysis were sucrose-impermeable. Spheroidal ghosts after complement lysis remained permeable for sucrose whereas spheroidal ghosts after nystatin lysis resealed after removal of the drug by washing. Biconcave ghosts produced by complement lysis were almost impermeable to sucrose initially and therefore responded to osmotic changes, but they became sucrose-permeable upon prolonged incubation at 37 degrees C. The rate of sucrose equilibration increased as the stability of the biconcave shape diminished with increasing numbers of C5b-9 complexes. At 850 C5b-9 complexes/ghost, the biconcave shape and impermeability for sucrose were completely lost. The results support the hypothesis that complement C5b-9 complexes, in addition to the interaction with the lipid bilayer, may interact with the cytoskeleton of the erythrocyte membrane.     

Association of a receptor and G-protein-regulated phospholipase C with the cytoskeleton.

Approximately 98% of turkey erythrocyte phospholipase C (PLC) is cytosolic and is released by hypotonic lysis of the cells and extensive washing of the resultant erythrocyte ghosts. Well washed turkey erythrocyte ghosts retain a fraction of tightly associated PLC, which is activated by the P2y-purinergic receptor and G-protein present in ghost membranes. The particulate PLC is sufficient to couple to all the available purinergic receptor-regulated G-protein. In contrast to ghosts, turkey erythrocyte plasma membrane preparations contain no detectable PLC. To investigate the subcellular location of the ghost-associated PLC, cytoskeletons were prepared by Triton X-100 extraction of turkey erythrocyte ghosts. The ghost-associated PLC was quantitatively recovered in cytoskeleton preparations. Cytoskeleton-associated PLC was solubilized by sodium cholate extraction, partially purified, and shown to reconstitute with PLC-free plasma membrane preparations in an agonist and guanine nucleotide-dependent fashion, indicating that the cytoskeleton-associated PLC is G-protein-regulated. Dissociation of erythrocyte ghost cytoskeletons with the actin-binding protein DNase 1 resulted in a dose-dependent inhibition of agonist and guanine nucleotide-stimulated PLC responses in ghosts and caused release of PLC from ghost or cytoskeleton preparations. These data demonstrate the specific association of a receptor and G-protein-regulated PLC with a component of the detergent-insoluble cytoskeleton and indicate that the integrity of the actin cytoskeleton is important for localization and effective coupling of PLC to the relevant G-protein.     

Immobilization of β-galactosidase retained in yeast: adhesion of the cells on a support

Summary Treatment of Kluyveromyces cells with several polycations partially neutralized their negative charge, even after washing; chitosan at 0.5 mg/ml rendered the zeta potential positive. Adhesion of the cells to various supports (glass, polycarbonate, polystyrene) was promoted by treating the cells or the support with chitosan; this confirms the importance of electrostatic interactions in the adhesion process. When the support was treated, a dense and regular monolayer of adhering cells was obtained; when the cells were treated the adhering layer was more heterogeneous due to floc formation. Cell ghosts of K. lactis were prepared by CHCl₃/ethanol permeabilization and glutaraldehyde treatment. Treating the ghosts with chitosan provided immobilization of 1.9 to 7.3×10⁶ ghosts per cm² according to the support; treating the support allowed to obtain 5.5 to 5.7×10⁶ ghosts per cm². The lactase activity of the immobilized ghosts was found to be comparable to that of ghosts in suspension, i.e. in the range of 0.9 to 2.2 U/mg dry cell weight, at 30°C, corresponding to 0.02 to 0.14 U/cm² of support. A test in microreactor with a lactase solution showed that 50% of the initial activity was still found, after about 25 days of continuous operation at 30°C.     

Intracellular distribution of subcellular organelles revealed by antibody against xyloglucan during cell cycle in tobacco BY-2 cells

Summary Immunofluorescence microscopy using an antibody against xyloglucan (XG) revealed its dynamics during the cell cycle. In interphase tobacco BY-2 cells, punctate and scattered fluorescence was observed throughout the cytoplasm. Colocalization of such signals with cortical microtubules (MTs) was clearly observed on the membrane ghosts. They were also associated and accumulated on MT bundles of the preprophase band. Treatment of protoplasts with cytochalasin B prior to the preparation of the ghosts had no effect on the pattern of anti-XG staining, while treatment with propyzamide caused the disappearance of the staining. These results suggest an association of Golgi apparatus and/or Golgi-derived vesicles with MTs. In metaphase cells, the staining was dispersed in the cytoplasm, except in the area occupied by the metaphase spindle. During anaphase, a broad fluorescence band appeared between daughter chromosomes and gradually concentrated at the equatorial plane before formation of the phragmoplast. At telophase, a bright line of fluorescence appeared at the equatorial plane corresponding to the position of the cell plate. The length of the line increased as cytokinesis proceeded. Thus, we showed that immunofluorescence microscopy using anti-XG antibody can be considered as a powerful tool for the analysis of Golgi apparatus and Golgi-derived vesicles containing XG.     

ISOLATION OF PLASMA MEMBRANE FRAGMENTS FROM HELA CELLS

A method for isolating plasma membrane fragments from HeLa cells is described. The procedure starts with the preparation of cell membrane "ghosts," obtained by gentle rupture of hypotonically swollen cells, evacuation of most of the cell contents by repeated washing, and isolation of the ghosts on a discontinuous sucrose density gradient. The ghosts are then treated by minimal sonication (5 sec) at pH 8.6, which causes the ghost membranes to pinch off into small vesicles but leaves any remaining larger intracellular particulates intact and separable by differential centrifugation. The ghost membrane vesicles are then subjected to isopycnic centrifugation on a 20–50% w/w continuous sucrose gradient in tris-magnesium buffer, pH 8.6. A band of morphologically homogeneous smooth vesicles, derived principally from plasma membrane, is recovered at 30–33% (peak density = 1.137). The plasma membrane fraction contained a Na-K-activated ATPase activity of 1.5 µmole Pi/hr per mg, 3% RNA, and 13.8% of the NADH-cytochrome c reductase activity of a heavier fraction from the same gradient which contained mitochondria and rough endoplasmic vesicles. The plasma membranes of viable HeLa cells were marked with ¹²⁵I-labeled horse antibody and followed through the isolation procedure. The specific antibody binding of the plasma membrane vesicle fraction was increased 49-fold over that of the original whole cells.     

Vesicle release from rat red cell ghosts and increased association of cell membrane proteins with cytoskeletons induced by cadmium

When rat red cell ghosts were incubated with 0.1-0.5 mM CdCl2 in 10 mM Tris-HCl (pH 7.4) at 37 degrees C for 30 min, they became irregular in shape and released small vesicles. The release of vesicles was dependent on the incubation temperature and Cd2+ concentration. The maximum release occurred at 37 degrees C in the presence of 0.2 mM Cd2+. The protein composition of Cd2+-induced vesicles was similar to that of the vesicles released from ATP-depleted red cells. Upon incubation with 0.1-0.2 mM Cd2+, more than 90% of the Cd2+ added to the incubation buffer was recovered in ghosts and 15-20% of the ghost Cd2+ was located on the cytoskeletons prepared by washing ghosts with 0.5% Triton X-100 solution containing 0.1 M KCl and 10 mM Tris-HCl (pH 7.4). Moreover, the cytoskeletons prepared from Cd2+-treated ghosts markedly contained cell membrane proteins, bands 2.1, 3, 4.2 and 4.5, and glycophorins. The association of bands 3 and 4.2 with cytoskeletons increased with increasing concentrations of Cd2+ added to the incubation buffer and saturated at 0.2 mM Cd2+. The solubilization of cytoskeletal proteins, bands 1, 2 and 5, from ghosts at low ionic strength was almost completely suppressed by preincubation of ghosts with 0.1 mM Cd2+. HgCl2, PbCl2 and ZnCl2 at 0.2 mM each also produced an increased association of cell membrane proteins with cytoskeletons, whereas CaCl2 and MgCl2 did not.     

Is an intact cytoskeleton required for red cell urea and water transport?

In order to determine the membrane protein(s) responsible for urea and water transport across the human red cell membrane, we planned to reconstitute purified membrane proteins into phosphatidylcholine vesicles. In preparatory experiments, we reconstituted a mixture of all of the red cell integral membrane proteins into phosphatidylcholine vesicles, but found that p-chloromercuribenzenesulfonate (pCMBS), which normally inhibits osmotic water permeability by approximately 90%, has no effect on this preparation. The preparation was also unable to transport urea at the high rates found in red cells, though glucose transport was normal. White ghosts, washed free of hemoglobin and resealed, also did not preserve normal urea and pCMBS-inhibitable water transport. One-step ghosts, prepared in Hepes buffer in a single-step procedure, without washing, retained normal urea and pCMBS-inhibitable water transport. Perturbations of the cytoskeleton in one-step ghosts, by removal of tropomyosin, or by severing the ankyrin link which binds band 3 to spectrin, caused the loss of urea and pCMBS-inhibitable water transport. These experiments suggest that an unperturbed cytoskeleton may be required for normal urea and pCMBS-inhibitable water transport. They also show that the pCMBS inhibition of water transport is dissociable from the water transport process and suggest a linkage between the pCMBS water transport inhibition site and the urea transport protein.     

False localization of acid phosphatase activity in the nuclear envelope and endoplasmic reticulum of peritoneal macrophages

Summary Acid phosphatase cytochemistry using lead salt methods was performed on rat peritoneal macrophages obtained by the intraperitoneal injection of dextran five days previously. Lead precipitate was present in the nuclear envelope, the rough endoplasmic reticulum, Golgi apparatus and lysosomes in about 50% of these cells. The formation of reaction product appeared to be substrate-specific and was sensitive to sodium fluoride in all these sites. However, only in the nuclear envelope, the rough endoplasmic reticulum and Golgi apparatus could lead salt precipitation be prevented by (a) omission of the washing procedure following the incubation step, (b) postincubation in a medium containing sodium fluoride, or (c) washing in buffer containing lead salt. It is concluded that precipitation of lead salt does not prove the presence of acid phosphatase activity in these organelles. The formation of precipitate in these sites is probably due to a local matrix effect, facilitated by the persistence of acid phosphatase activity in the lysosomes and a suboptimal trapping efficiency of phosphate ions during the washing procedure which follows in the incubation step.     

Fusion of cell ghosts with intact cells in Dictyostelium discoideum: Differential response of opposite mating-type cells

The sexual cycle of Dictyostelium discoideum is initiated by the fusion of cells that are of opposite mating types (e.g. NC4- and HM1-type cells). Cells grown in light on agar plates are not capable of sexual cell fusion, but become capable when cultured in the dark in a liquid medium. Cells in the incapable state are called fusion-incompetent cells, and cells in the latter state, fusion-competent cells. To gain some understanding of the mechanism of cell fusion, cell ghosts prepared by freeze-thawing intact cells were incubated with intact cells. The cell ghosts killed the intact cells by directly fusing with them, the extent of fusion depending on the particular strains employed and the fusion-competency of the intact cells and of the cells from which the cell ghosts had been prepared. A detailed examination revealed that fusion-competent NC4 cells were always more easily killed by cell ghosts than fusion-incompetent NC4 cells. It also became apparent that cell ghosts prepared from fusion-competent NC4 cells killed all cell types far more efficiently than did those prepared from fusion-incompetent NC4 cells. However, fusion-competent and fusion-incompetent HM1 cells were equally sensitive to cell ghosts, and cell ghosts prepared from fusion-competent HM1 cells had the same ability to kill as those prepared from fusion-incompetent HM1 cells. From these findings, it thus appears that opposite mating-type cells have distinct membrane properties related to sexual cell fusion.     

Extraction of membrane proteins in low ionic strengths]

Hemoglobin and the low molecular weight proteins 8 and 9 are extracted from ghosts during low ionic washing after the hypotonic hemolysis of erythrocytes. Furthermore, a loss of the proteins 4.5 and 7 was observed. The protein patterns of ghosts after isotonic hemolysis by freezing and thawing resemble the ghost protein patterns after hypotonic hemolysis and incomplete deprivation of Hb. Many if not all membrane proteins are eluted by repeated incubations of the ghosts in solutions of low ionic strength in the presence of EDTA. The spectrins, the proteins 5, 4.5, 7 and residual Hb are extracted preferentially. A selective extraction of the spectrins and the protein 5 is not detectable under these conditions. Often the spectrin bands are subdivided following low ionic incubation.     

The role of ATP in swelling-stimulated K-Cl cotransport in human red cell ghosts. Phosphorylation-dephosphorylation events are not in the signal transduction pathway

Volume-sensitive K-Cl cotransport occurs in red blood cells of many species. In intact cells, activation of K-Cl cotransport by swelling requires dephosphorylation of some cell protein, but maximal activity requires the presence of intracellular ATP. We have examined the relation between K-Cl cotransport activity and ATP in ghosts prepared from human red blood cells. K-Cl cotransport activity in swollen ghosts increased by ATP, and the increase requires Mg so that it almost certainly results from the phosphorylation of some membrane component. However, even in ATP-free ghosts residual volume-sensitive K-Cl cotransport can be demonstrated. This residual cotransport in ATP-free ghosts is greater in the presence of vanadate, a tyrosyl phosphatase inhibitor, and in ghosts that contain ATP cotransport is reduced by genistein, a tyrosyl kinase inhibitor. Okadaic acid, an inhibitor of serine and threonine phosphatases, inhibits K-Cl cotransport in ghosts as it does in intact cells. Experiments in which ghosts were preexposed to okadaic acid showed that the protein dephosphorylation that permits K-Cl cotransport can proceed to completion before the ghosts are swollen and K transport measured and therefore dephosphorylation is not a response to ghost swelling. In experiments with ATP-free ghosts we found that phosphorylation is not necessary to increase the cotransport rate when shrunken ghosts are swollen, nor is rephosphorylation necessary to decrease the cotransport rate when swollen ghosts are shrunken. Cotransport is greater in swollen than in shrunken ghosts even when the swollen and shrunken ghosts have the same concentration of cytoplasmic solutes. We conclude that, although phosphorylation and dephosphorylation modify the activity of the cotransporter in swollen and in shrunken ghosts, neither of these processes nor any other known messenger is involved in signal transduction between the cell volume sensor and the cotransporter as originally proposed by Jennings and Al- Rohil (Jennings, M. L., and N. Al-Rohil. 1990. Journal of General Physiology. 95: 1021-1040).     

A comparison of intact human red blood cells and resealed and leaky ghosts with respect to their interactions with surface labelling agents and proteolytic enzymes

Resealed ghosts and intact red blood cells were directly compared with respect to their interactions with surface probes and to digestion by pronase. The amount and pattern of labelling of surface proteins by 4.4′-diisothiocyano-2,2′-stilbene disulfonic acid (DIDS) and by pyridoxal phosphate-borohydride (as seen after sodium dodecylsulfate/acrylamide gel electrophoresis) was substantially the same in cells and resealed ghosts under conditions in which a relatively small change would be apparent. In each membrane system, DIDS labels a protein component of apparent molecular weight 95 000 and pyridoxal phosphate labels the same protein plus three glycoprotein components. The sensitivity of surface proteins and of DIDS and pyridoxal phosphate-labelled sites to pronase was also similar in the cells and resealed ghosts. The glycoproteins were digested, in each case, and the 95 000 (molecular weight) protein was largely split into two portions of apparent molecular weights 65 000 and 35 000, with both portions containing DIDS and pyridoxal phosphate binding sites. The pattern of labelling of “leaky” ghosts by pyridoxal phosphate in the presence of hemoglobin was similar to the labelling of intact cells, provided that the pyridoxal phosphate was present on both the outside and inside of the cells. Virtually all of the major protein components visible by staining on acrylamide gels were labelled. It is concluded that none of the probes could detect any substatial differences in reactivity of proteins of the outer surface of the membrane of the ghosts as compared to the cells and that no irreversible changes in membrane protein conformation or arrangement occur as a consequence of lysis and resealing of ghosts, that are detectable by the reported procedures.     

Transformation of L cells with virus thymidine kinase genes introduced by red cell-mediated microinjection

Fusion of red cell ghosts containing foreign materials with cells results in the introduction of the materials into the cells (red cell-mediated microinjection). Until now, 'two-step dialysis' has mainly been used for trapping proteins in the ghosts. Large-sized materials such as DNA, however, are rarely trapped in the ghosts, since the holes in the red cell membrane caused by osmotic shock are too small for such materials to pass through. In this study, we improved the trapping technique. Some of the Hind III fragments of lambda phage DNA as well as proteins could be trapped in the ghosts when the mixture of these materials and red cells were frozen at -80 degrees C for a short period followed by quick thawing. Red cell-mediated microinjection using ghosts containing plasmid pBR322 linked with a Herpes simplex viral thymidine kinase (tk) gene brought about transformation of tk-defective L cells, the efficiency of transformation was 1 out of 20 000-60 000 cells fused with the ghosts.     

Quantitative introduction of a given macromolecule into cells by fusion with erythrocyte ghosts using a fluorescence activated cell sorter.

FITC-conjugated bovine serum albumin (FITC-BSA) molecules were quantitatively introduced into human erythrocyte ghosts by gradual hemolysis. When the ghosts and L cells were fused with UV-inactivated HVJ (Sendai virus), FITC-BSA was introduced into the cytoplasm of the L cells and fluorescence could be observed inthe cells with a fluorescence microscope. A mixture of L cells and ghosts was introduced into a fluorescence activated cell sorter (FACS), which could separate the mononuclear cells on the basis of their light-scattering profile. Four distinct populations of mononuclear cells were found by fluorescence analysis. These populations were separated from the cell mixture and found to correspond to cells fused with one, two and three ghosts and unfused cells. After separation, the cells from each population could form colonies in culture. As a given macromolecule can be quantitatively introduced into erythrocyte ghosts with the FITC-BSA, after fusion of these ghosts with cells, this sorting method is useful for separating cells containing a definite number of macromolecules.     

Hemoglobin-depleted human erythrocyte ghosts: Characterization of morphology and transport functions

Summary A method of preparing hemoglobin-depleted resealed ghosts with an extremely low hemoglobin content is described. The membrane morphology, the crossed immunoelectrophoresis pattern of the membrane proteins, and the transport function of these ghosts have been examined.Electron microscopic examination of the ghosts on hydrophilic as well as hydrophobic grid surfaces revealed a faint filamentous network (spectrin) associated with the membrane. The ghosts were found to have permeabilities towards small polar molecules (water and mannitol) and ions (chloride, sodium, and potassium) which are quantitatively very close to those of intact red cells.It is concluded that white ghosts prepared by the present method are well suited for simultaneous studies of morphology, membrane biochemistry, and membrane transport functions.     

A quantitative study of ultramicroinjection of macromolecules into animal cells.

Improvements in the technique of ultramicroinjection of macromolecules into animal cells are described. The method is based on the Sendai virus-induced fusion of animal cells with erythrocyte ghosts containing trapped macromolecules. Fusion of hepatoma tissue culture (HTC) cells with ghosts prepared by hemolysis of erythrocytes in the presence of cytochrome C is much more efficient than fusion with ghosts prepared in the presence of bovine serum albumin (BSA) as in previous investigations. La+++ is more fficient in promoting fusion and less toxic to cells than Mn++, which was used previously. Thus in all subsequent experiments, erythrocytes were hemolyzed in the presence of cytochrome C plus other macromolecules to be trapped, and the resultant ghosts fused in the presence of La+++. The percentage of HTC cells which fused with ghosts reached 80% in many experiments. Ghosts containing 125I-BSA were used to measure the number of BSA molecules injected into HTC cells. About 10(6) BSA molecules were injected per fused cell. The overall efficiency of injection was low (about 0.02% of the starting material).     

Bacterial ghosts are an efficient delivery system for DNA vaccines

Mass implementation of DNA vaccines is hindered by the requirement of high plasmid dosages and poor immunogenicity. We evaluated the capacity of Mannheimia haemolytica ghosts as delivery system for DNA vaccines. In vitro studies showed that bacterial ghosts loaded with a plasmid carrying the green fluorescent protein-encoding gene (pEGFP-N1) are efficiently taken up by APC, thereby leading to high transfection rates (52-60%). Vaccination studies demonstrated that ghost-mediated delivery by intradermal or i.m. route of a eukaryotic expression plasmid containing the gene coding for beta-galactosidase under the control of the CMV immediate early gene promoter (pCMVbeta) stimulates more efficient Ag-specific humoral and cellular (CD4(+) and CD8(+)) immune responses than naked DNA in BALB/c mice. The use of ghosts also allows modulating the major Th response from a mixed Th1/Th2 to a more dominant Th2 pattern. Intravenous immunization with dendritic cells loaded ex vivo with pCMVbeta-containing ghosts also resulted in the elicitation of beta-galactosidase-specific responses. This suggests that dendritic cells play an important role in the stimulation of immune responses when bacterial ghosts are used as a DNA delivery system. Bacterial ghosts not only target the DNA vaccine construct to APC, but also provide a strong danger signal, acting as natural adjuvants, thereby promoting efficient maturation and activation of dendritic cells. Thus, bacterial ghosts constitute a promising technology platform for the development of more efficient DNA vaccines.     

Effect of carbonylcyanide m-chlorophenylhydrazone on the calcium-stimulated ATPase activity of erythrocyteghosts.

Incubation of etythrocyte ghosts with carbonylcyanide m-chlorophenyl-hydrazone (CCCP) plus Ca-2+ resulted in inactivation of the Ca-2+ -stimulated ATPase activity. Omission of Ca-2+ or lowering of the temperature below 25 degrees C eliminated the inhibitory effect, as also did the presence of ATP during the incubation. On the other hand, the addition of beta-mercaptoethanol did not influence the Ca-2+ -dependent inhibition by CCCP. Compared with the level of CCCP which uncouples oxidative phosphorylation, a rather high level (0.5 mM) of CCCP was required to inhibit the ATPase activity in ghosts. However, once the inhibition had been accomplished, almost all of the CCCP could be removed from the ghost membrane by washing with a Ca-2+ -containing solution, without affecting the inhibition of ATPase. If ethylene-glycol-bis(beta-aminoethyl ether)-N,N'-tetraacetic acid was included in the washing medium, the inhibition of ATPase was nearly completely reversed by washing. The results indicate that only the Ca-2+ -stimulated, Mg-2+ -ATPase was inhibited by 0.5 mM CCCP, while the remaining components of the ATPase activity were slightly inhibited by higher levels of the uncoupler. Low levels of CCCP (0.1 mM) stimulated the Mg-2+ -ATPase slightly. CCCP was much more specific for the Ca-2+ -stimulated ATPases than N-(1-naphthyl)maleimide, an unusually effective sulfhydryl reagent, and the requirement of Ca-2+ for inactivation was also quite specific.     

Improved stop-flow apparatus to measure permeability of human red cells and ghosts

An improved stop-flow apparatus has been designed and constructed to measure the permeability characteristics of human red cells, which can be inferred from the time course of red cell volume changes following a sudden change in cellular environment produced by a rapid mixing device. The improved apparatus is directly coupled to a computer which automates thesubtraction and averaging procedures that have been developed to minimize the noise generated in the system by the cessation of red cell forward motion when the flow is suddenly stopped. Real time data acquisition also makes it possible to increase the number of data points by an order of magnitude, thus improving accuracy significantly. The apparatus has been tested by measurements of the human red cel hydraulic permeability coefficient. Data are presented to validate the subtraction procedure. Experiments have also been carried out on red cell ghosts which indicate that the hydraulic conductivity of the ghost is similar to that of the undisturbed red cell.