Activation of Alpha Chymotrypsin by Three Phase Partitioning Is Accompanied by Aggregation

Precipitation of alpha chymotrypsin in the simultaneous presence of ammonium sulphate and t-butanol (three phase partitioning) resulted in preparations which showed self aggregation of the enzyme molecules. Precipitation with increasing amounts of ammonium sulphate led to increasing size of aggregates. While light scattering estimated the hydrodynamic diameter of these aggregates in the range of 242–1124 nm; Nanoparticle tracking analysis (NTA) gave the value as 130–462 nm. Scanning electron microscopy and gel filtration on Sephadex G-200 showed extensive aggregation in these preparations. Transmission electron microscopy showed that the aggregates had irregular shapes. All the aggregates had about 3× higher catalytic activity than the native enzyme. These aggregates did not differ in λ_max of fluorescence emission which was around 340 nm. However, all the aggregates showed higher fluorescence emission intensity. Far-UV and near-UV circular dichroism also showed no significant structural changes as compared to the native molecule. Interestingly, HPLC gel filtration (on a hydroxylated silica column) gave 14 nm as the diameter for all preparations. Light scattering of preparations in the presence of 10% ethylene glycol also dissociated the aggregates to monomers of 14 nm. Both these results indicated that hydrophobic interactions were the driving force behind this aggregation. These results indicate: (1) Even without any major structural change, three phase partitioning led to protein molecules becoming highly prone to aggregation. (2) Different methods gave widely different estimates of sizes of aggregates. It was however possible to reconcile the data obtained with various approaches. (3) The nature of the gel filtration column is crucial and use of this technique for refolding and studying aggregation needs a rethink.     

Multivalent Binding of a Ligand-Coated Particle: Role of Shape,Size, and Ligand Heterogeneity

We utilize a multiscale modeling framework to study the effect of shape, size, and ligand composition on the efficacy of binding of a ligand-coated particle to a substrate functionalized with the target receptors. First, we show how molecular dynamics along with steered molecular dynamics calculations can be used to accurately parameterize the molecular-binding free energy and the effective spring constant for a receptor-ligand pair. We demonstrate this for two ligands that bind to the ${\alpha }_5{\beta }_1$-domain of integrin. Next, we show how these effective potentials can be used to build computational models at the meso- and continuum-scales. These models incorporate the molecular nature of the receptor-ligand interactions and yet provide an inexpensive route to study the multivalent interaction of receptors and ligands through the construction of Bell potentials customized to the molecular identities. We quantify the binding efficacy of the ligand-coated-particle in terms of its multivalency, binding free-energy landscape, and the losses in the configurational entropies. We show that 1) the binding avidity for particle sizes less than 350 nm is set by the competition between the enthalpic and entropic contributions, whereas that for sizes above 350 nm is dominated by the enthalpy of binding; 2) anisotropic particles display higher levels of multivalent binding compared to those of spherical particles; and 3) variations in ligand composition can alter binding avidity without altering the average multivalency. The methods and results presented here have wide applications in the rational design of functionalized carriers and also in understanding cell adhesion.     

Cytoskeleton Modification and Cholesterol Depletion Affect Membrane Properties and Caveolae Positioning of CHO Cells

The formation of protrusions is necessary for numerous biological processes. It involves extension of the plasma membrane, and the force needed for this is provided by the actin cytoskeleton. Tether pulling with optical tweezers can mimic the formation of a protrusion, so we used this method to investigate the effects of modifying not only actin (with latrunculin A) but also microtubules (with nocodazole) and the plasma membrane itself (with methyl-β-cyclodextrin) on the Chinese hamster ovary cell membrane. After these modifications, the membrane reservoir was supposed to redistribute. Caveolae constitute a small part of the reservoir, so the redistribution of caveolar proteins such as caveolin-1 and cavin-1 that represents caveolae per se was assessed. The main findings concerning protrusion force and membrane reservoir availability were as follows: (1) they correlated inversely, (2) their values underwent the greatest change after microtubule disruption, and (3) membrane composition had a major influence on the parameters studied. F-actin disruption and cholesterol depletion decreased, and microtubule disruption increased the amount of the caveolar proteins (caveolae). Caveolae presented just an example of the membrane reservoir, and from our findings, we suppose that the perturbations caused were too large to be related to caveolae redistribution alone. The integrity of the cytoskeleton and plasma membrane composition are important factors in the formation of protrusions and in determining the availability and distribution of the membrane reservoir.     

Membrane Cholesterol Depletion and K+ Transport in High and Low Potassium Sheep Red Cells

The effect of cholesterol depletion on potassium tracer fluxes was studied in sheep red cells. Removal by the plasma incubation method (5, 12, 30) of approximately 31 and 34% membrane cholesterol from high-potassium (HK) and low-potassium (LK) sheep red cells, respectively, did not induce significant changes in the steady-state cation composition of these cells nor in their passive (leak) and active (pump) K⁺ influxes. In cholesterol-depleted LK sheep red cells, there was no impairment nor augmentation of the L_p an tibody stimulated K⁺ pump flux and L₁-antibody-mediated reduction of K⁺ leak flux indicating that the removed cholesterol does not contribute to the activity of the L_p and L₁ antigens.     

Cholesterol-Dependent Bending Energy Is Important in Cholesterol Distribution of the Plasma Membrane

We consider the plasma membrane that contains a cholesterol molar fraction of 0.4 and ask how that cholesterol is distributed between the two leaves. Because of the rapid flip-flop of cholesterol between leaves, we assume that its distribution is determined by the equality of its chemical potentials in the two leaves. When we consider only the contributions of entropy and interactions to the cholesterol chemical potential in our model system, we find, not surprisingly, that the cholesterol is mostly in the outer leaf because of the strong attraction between cholesterol and sphingomyelin (SM), which is predominantly in that leaf. We find 72% there. We then include the contribution from the bending energy in each leaf that must be overcome to join the leaves in a flat bilayer. The product of bending modulus and spontaneous curvature is obtained from simulation. We find that the addition of cholesterol to the outer leaf reduces the spontaneous curvature, which is initially positive, until it passes through zero when the molar fraction of cholesterol in the outer leaf is 0.28. Additional cholesterol is driven toward the inner leaf by the sphingomyelin phosphatidylcholine mixture. This is resisted by the bending energy contribution to the inner leaf. We find, again by simulation, that the addition of cholesterol monotonically increases the magnitude of the spontaneous curvature of the inner leaf, which is negative. This increases its bending energy. We conclude that, as a result of these competing effects, the percentage of cholesterol in the outer leaf is reduced to ∼63 ± 6%.     

Esterified Cholesterol Is Highly Localized to Bruch's Membrane, as Revealed by Lipid Histochemistry in Wholemounts of Human Choroid

Accumulation of neutral lipids in Bruch''s membrane (BrM) is a major age change in human retina and contributes to the formation of extracellular lesions associated with age-related macular degeneration. We developed a BrM–choroid wholemounting technique suitable for reliable staining and evaluated different fluorescent lipid dyes for topographic semiquantitative analysis of BrM lipids. Thin BrM–choroid complexes with partially stripped choroid from 10 aged donor eyes were prepared with an optimized wholemounting technique. Preparation quality was monitored by examining 1-μm-thick sections of representative samples. The staining patterns of Nile Red, BODIPY 493/503, filipin for unesterified cholesterol (UC-F), filipin for esterified cholesterol (EC-F), and Oil Red O in wholemounts were compared with their staining patterns in chorioretinal sections, using wide-field epi-fluorescence microscopy. Wholemounts exhibited optimal flatness on the BrM side. Reduced tissue thickness allowed reliable dye penetration and staining of BrM. Only EC-F was with high specificity localized to BrM and demonstrated an intense and distinct granular staining pattern not previously appreciated in chorioretinal sections. All other lipid dyes also stained choroidal or retinal tissue intensely. No dye provided perfect characteristics in regard to representing all neutral lipid classes present in BrM or to fluorescence intensity. Nevertheless, only EC-F was highly localized to BrM with a specific granular pattern. Because direct assays indicate that esterified cholesterol is abundantly present in BrM, we consider EC-F the most valuable choice for analyzing neutral lipid deposits in human BrM. (J Histochem Cytochem 57:731–739, 2009)     

Leishmania donovani Infection Enhances Lateral Mobility of Macrophage Membrane Protein Which Is Reversed by Liposomal Cholesterol

BackgroundThe protozoan parasite Leishmania donovani (LD) reduces cellular cholesterol of the host possibly for its own benefit. Cholesterol is mostly present in the specialized compartment of the plasma membrane. The relation between mobility of membrane proteins and cholesterol depletion from membrane continues to be an important issue. The notion that leishmania infection alters the mobility of membrane proteins stems from our previous study where we showed that the distance between subunits of IFNγ receptor (R1 and R2) on the cell surface of LD infected cell is increased, but is restored to normal by liposomal cholesterol treatment.Conclusions/SignificancesTo our knowledge this is the first direct demonstration that LD parasites during their intracellular life cycle increases lateral mobility of membrane proteins and decreases F-actin level in infected macrophages. Such defects may contribute to ineffective intracellular signaling and other cellular functions.     

Membrane Cholesterol,Tumorigenesis, and the Biochemical Phenotype of Neoplasia

The loss of sensitivity to feedback control of lipid synthesis in tumors is especially manifest with regard to cholesterol biosynthesis. This phenomenon has been documented in a wide variety of cells soon after exposure to a carcinogenic insult, and results in the progressive enrichment of diverse cellular membranes with cholesterol during the tumorigenic process. By the time a tumor is identifiable histopathologically, such membrane enrichment with cholesterol can lead to significant changes in the activities of membrane-affiliated enzymes. This, in turn, can effect a wide variety of altered metabolic patterns within the afflicted cell, thereby promoting altered cell function. Some of the altered metabolic patterns in tumors that are consistent with events leading to cholesterol-enriched membranes include increased glutamate and glutamine oxidation in support of tumor bioenergetics; reduced rates of glucose oxidation via respiration together with enhanced levels of aerobic glycolysis; diminished urea cycle activity in hepatomas; increased polyamine accumulation; shifts in the pattern of reducing equivalent activities in mitochondria; and in the tumor-burdened host, the exfoliation of cholesterol-rich microvesicles from the tumor cell plasma membrane, which decoy the host's immune surveillance away from the tumor itself. The obligatory presence of cholesterol in cell membranes thus acquires a greater significance, not only to normal cell dynamics, but to metabolic phenomena that are characteristic of the neoplastic phenotype.     

Biophysical Interactions of Membrane Anionic Phospholipids with pH, Calcium and Auxins

A series of phospholipid monolayers—phosphatidylinositol,phosphatidylserine and phosphatidyl 4, 5-bisphosphate, possessingan increasingly negative headgroup potential, were spread andcompressed over aqueous sub-phases and, under varying conditions,assayed for changes in surface parameters. These included surfacetension and degree of molecular compression expressed by surfacepressure/molecular area isotherms. Experimentation employingWilhelmy-Du Nouy tensio-metry and the Langmuir-trough compressionprocedure indicated that with increasing electrostatic Ca²⁺cross-linking, this being a direct function of Ca²⁺ concentrationin the aqueous sub-phase, there was a concomitant increase insurface tension, i.e. monolayer condensation. This effect wasgreater with the more negatively charged phospholipids and wasmore pronounced at pH 7–0 than at pH 4–5. Applicationof the auxins indoleacetic acid, indolebutyric acid and naphthaleneaceticacid (which presumably act as proton donors which may dislodgecross-linking Ca²⁺ ) causes a significant decrease in surfacetension when applied at physiological Ca²⁺ concentrations. Asevidenced by surface pressure/molecular area isotherms, Ca²⁺increases monolayer rigidification and decreases phosphatidyl4, 5-bisphosphate molecular area. The auxins tested possessan opposite and de-ngidifying effect as indicated by increaseof the phospholipid’s molecular area and by the observedconsiderable lowering of monolayer collapse pressures. The surfactiveeffectivity of the auxins increased with decreasing sub-phaseCa²⁺ concentration in the 10^–4 to 10^–6mol dm^–3range. These data collectively suggest that surface-associatedbiophysical changes should be taken into account during interpretationof transmembrane hormonal signal transduction. Key words: Membrane phospholipids, monolayers, surface properties     

Linear Plasmid SLP2 Is Maintained by Partitioning,Intrahyphal Spread,and Conjugal Transfer in Streptomyces

Low-copy-number plasmids generally encode a partitioning system to ensure proper segregation after replication. Little is known about partitioning of linear plasmids in Streptomyces. SLP2 is a 50-kb low-copy-number linear plasmid in Streptomyces lividans, which contains a typical parAB partitioning operon. In S. lividans and Streptomyces coelicolor, a parAB deletion resulted in moderate plasmid loss and growth retardation of colonies. The latter was caused by conjugal transfer from plasmid-containing hyphae to plasmidless hyphae. Deletion of the transfer (traB) gene eliminated conjugal transfer, lessened the growth retardation of colonies, and increased plasmid loss through sporulation cycles. The additional deletion of an intrahyphal spread gene (spd1) caused almost complete plasmid loss in a sporulation cycle and eliminated all growth retardation. Moreover, deletion of spd1 alone severely reduced conjugal transfer and stability of SLP2 in S. coelicolor M145 but had no effect on S. lividans TK64. These results revealed the following three systems for SLP2 maintenance: partitioning and spread for moving the plasmid DNA along the hyphae and into spores and conjugal transfer for rescuing plasmidless hyphae. In S. lividans, both spread and partitioning appear to overlap functionally, but in S. coelicolor, spread appears to play the main role.Soil bacteria of the genus Streptomyces possess terminal protein (TP)-capped linear chromosomes (23) and often harbor one or more linear plasmids with similar structures (29). Like their circular counterparts, many of these linear plasmids mediate conjugation and transfer of themselves and the chromosomes (reviewed in references 12 and 13).Conjugation in Streptomyces often produces a unique transfer-related phenotype known as “pocking” or “lethal zygosis” (4, 5). On certain solid media, transfer of a conjugative plasmid from donor to recipient leads to a growth inhibition zone (“pock”), reflecting retarded growth and development of recipient mycelia. Different plasmids display distinct pock morphology.Streptomyces plasmids often contain only a single transfer gene (tra or kil) for mobilization of the plasmid from donor to recipient hyphae. The Streptomyces TraB proteins resemble the SpoIIIE protein of Bacillus subtilis, which translocates double-stranded chromosomal DNA during prespore formation (35). The genetic study of Possoz et al. (27) suggests that pSAM2, a circular Streptomyces plasmid, is transferred in double-stranded form. In support of this, Reuther et al. (28) showed that the TraB protein of the circular Streptomyces plasmid pSVH1 binds specifically to a 14-bp direct repeat downstream of the traB gene without any detectable nicking activity, which is required for conjugal transfer of plasmid DNA in single-stranded form via rolling circle replication.Overexpression of the Streptomyces traB genes is lethal to the host (hence the name kil in some cases), and the presence of kil override (kor) genes is necessary for viability of the host (17). Mutations in the tra or kil gene completely abolish conjugal transfer and pocking. The mechanism of killing by traB or kil is unknown.After the entry into the recipient mycelium, spreading of the incoming plasmid along the recipient hyphae has been proposed to rely on the plasmid-carried spread (spd) genes. The conjugative plasmids of Streptomyces usually contain two or more spd genes, which encode small hydrophobic proteins that often do not show extensive sequence similarity to one another or to other proteins in the database (9). Inactivation of a single spd gene is sufficient to cause a reduction in pock sizes (16, 19), and it was therefore postulated that the spd genes promote migration of plasmid copies inside the recipient mycelium (19).Recently, Tiffert et al. (33) showed that overexpression of Spd2, like that of TraB, was also lethal to the host. They also demonstrated that in vitro, a spread gene product, SpdB2, formed oligomers and interacted with the TraB protein. Those authors proposed that TraB, SpdB2, and other spread proteins formed a channel at the septal cross walls for intrahyphal spread of the plasmid DNA. Involvement of TraB in spreading has also been suggested by Kataoka et al. (16), Kosono et al. (21), and Pettis and Cohen (26). This postulated involvement of Spd proteins and TraB in intrahyphal spread has, however, not yet been experimentally demonstrated.During vegetative growth, distribution of low-copy-number plasmids in dividing bacterial cells usually involves a functional partitioning system. In Streptomyces, proper postreplicational partitioning of low-copy-number plasmids is important during the development of haploid spores from aerial hyphae to avoid plasmid loss in the subcultures, and this task is generally assumed to rely on a parAB operon, which is also used in the partitioning of many other bacterial chromosomes and plasmids (reviewed in reference 8). ParB is a DNA binding protein, which binds specifically to one or more centromere-like parS sites. ParA, a membrane-associated ATPase, is recruited by ParB to the parS site and forms a cytoskeletal structure required for the symmetric movement of the ParB-parS complex during partitioning. The involvement of conjugal transfer, spread, and partitioning in the movement of plasmids in a Streptomyces colony is depicted in Fig. ​Fig.1A1A.Open in a separate windowFIG. 1.Models for partitioning, transferring, and spreading of SLP2 and their interactions in Streptomyces. (A) During vegetative growth of the mycelium, the spread of the low-copy-number plasmid SLP2 (dumbbells) along the substrate and into the aerial hyphae is aided by both the parAB partitioning system (Par) and the spread system (Spd). The proper partitioning of the plasmids into the haploid spores also involves the partitioning system. Gray blotches represent the chromosomes. (B) Involvement of conjugal transfer (Tra) and Spd systems in compensation of SLP2 loss and growth retardation caused by the ΔparAB mutation. Partitioning defect during formation of spores from aerial hyphae (top) results in the absence of SLP2tsrΔpar plasmids in some spores (path a). Colonies developed from the plasmidless spores would be of normal size and produce Thio^s spores. If a plasmid-containing spore suffers severe plasmid loss during colony development (path b), extensive Tra-mediated interhyphal transfer and Spd-mediated intrahyphal transfer of the plasmid (arrows) would result in acute growth retardation, and the spores produced would contain SLP2tsrΔpar and be Thio^r. If the plasmid suffers no or little loss during colony formation (path c), conjugal transfer and growth retardation would be minimal, and most of the spores produced would be Thio^r.One or more parAB operons are found on low-copy-number plasmids in Streptomyces, the linear SLP2 plasmid in Streptomyces lividans (15), and the linear SCP1 plasmid (2) and the circular plasmid SCP2 (11) in Streptomyces coelicolor, all of which are stably maintained in their hosts. Of these, the parAB system of SCP2 has been demonstrated to be important for stable maintenance of SCP2 through sporulation cycles in S. coelicolor (11). Supposedly, the parAB system in the linear plasmids plays the same functional role.The linear chromosome of Streptomyces also possesses a parAB operon. In S. coelicolor, about 20 parS sequences are clustered within 520 kb of oriC (3, 20). Disruption of the parAB operon on the S. coelicolor chromosome resulted in 13% of anucleate spores (20). Interestingly, this defect can be suppressed by the presence of an integrated form of the linear plasmid SCP1 (the NF state), which contains two parAB homologs (2). Other than these studies, little is known about the mechanisms of stable maintenance of low-copy-number plasmids in Streptomyces, especially the linear ones.In this study, we initiated a study of the genetic controls of SLP2 stability. SLP2, a 50-kb linear plasmid originally identified in S. lividans 1326 (6, 15), may be conjugally transferred to and stably maintained in various other species (14). It contains 43 putative genes, including 3 involved in conjugation, spd1 (SLP2.18), traB (SLP2.19), and spd2 (SLP2.26), and a putative par operon consisting of parA (SLP2.30c) and parB (SLP2.29c) (15). We discovered that deletion of parAB (ΔparAB) causes only a mild defect in maintenance of SLP2 and surprisingly resulted in growth retardation of SLP2-containing colonies. We present evidence showing that the mild effect of the ΔparAB mutation was due to recovery of the plasmid through traB-mediated conjugal transfer from plasmid-containing to plasmidless hyphae during growth, and the growth retardation was caused by the interhyphal conjugal transfer and spd1-mediated intrahyphal spread. In addition, the intrahyphal spread function is also important for SLP2 maintenance during vegetative growth. It appears to overlap fully with the partitioning function in S. lividans (the original host) but dominates over the partitioning function in S. coelicolor, such that deletion of spd1 caused little effect on S. lividans but severely reduced the stability and conjugal transfer efficiency of SLP2 in S. coelicolor.In contrast to unicellular bacteria, the involvement of multiple mechanisms for plasmid maintenance reflects the demand for efficient movement of plasmid DNA along the substrate and aerial hyphae before partitioning into the spores during the relatively complex life cycle of Streptomyces.     

Selective and Synergistic Activity of L-S,R-Buthionine Sulfoximine on Malignant Melanoma Is Accompanied by Decreased Expression of Glutathione-S-Transferase

L-buthionine-S,R-sulfoximine (BSO) selectively inhibits glutathione (GSH) synthesis. Malignant melanoma may be uniquely dependent on GSH and its linked enzymes, glutathione S-transferase (GST) and GSH-peroxidase, for metabolism of reactive orthoquinones and peroxides produced during melanin synthesis. We compared the in vitro effects of BSO on melanoma cell lines and fresh melanoma specimens (n = 118) with breast and ovarian cell lines and solid tumors (n = 244). IC₅₀ values (μM) for BSO on melanoma, breast and ovarian tumor specimens were 1.9, 8.6, and 29, respectively. The IC₉₀ for melanoma was 25.5 μM, a level 20-fold lower than steady state levels achieved clinically. The sensitivity of individual specimens of melanoma correlated with their melanin content (r = 0.63). BSO synergistically enhanced BCNU activity against melanoma cell lines and human tumors. We followed GSH levels, GST enzyme activity, GST isoenzyme profiles and mRNA levels after BSO. BSO (50 μM) treatment for 48 hr resulted in a 95% decrease in ZAZ and M14 melanoma cell line GSH levels, and a 60% decrease in GST enzyme activity. GST-μ. protein and mRNA levels were significantly reduced in both cell lines. GST expression was unaffected. These data suggest that BSO action on melanoma may be related to GSH depletion, diminishing the capacity to scavenge toxic metabolites produced during melanin synthesis. We report here for the first time that BSO enhancement of alkylator action may be related in part to down regulation of GST. BSO may be a clinically useful adjunct in the treatment of malignant melanoma.     

The Evolution of Selfing Is Accompanied by Reduced Efficacy of Selection and Purging of Deleterious Mutations

The transition from outcrossing to selfing is predicted to reduce the genome-wide efficacy of selection because of the lower effective population size (N_e) that accompanies this change in mating system. However, strongly recessive deleterious mutations exposed in the homozygous backgrounds of selfers should be under strong purifying selection. Here, we examine estimates of the distribution of fitness effects (DFE) and changes in the magnitude of effective selection coefficients (N_es) acting on mutations during the transition from outcrossing to selfing. Using forward simulations, we investigated the ability of a DFE inference approach to detect the joint influence of mating system and the dominance of deleterious mutations on selection efficacy. We investigated predictions from our simulations in the annual plant Eichhornia paniculata, in which selfing has evolved from outcrossing on multiple occasions. We used range-wide sampling to generate population genomic datasets and identified nonsynonymous and synonymous polymorphisms segregating in outcrossing and selfing populations. We found that the transition to selfing was accompanied by a change in the DFE, with a larger fraction of effectively neutral sites (N_es < 1), a result consistent with the effects of reduced N_e in selfers. Moreover, an increased proportion of sites in selfers were under strong purifying selection (N_es > 100), and simulations suggest that this is due to the exposure of recessive deleterious mutations. We conclude that the transition to selfing has been accompanied by the genome-wide influences of reduced N_e and strong purifying selection against deleterious recessive mutations, an example of purging at the molecular level.     

Cholesterol Depletion Mimics the Effect of Cytoskeletal Destabilization on Membrane Dynamics of the Serotonin1A Receptor: A zFCS Study

Single-point fluorescence correlation spectroscopy (FCS) of membrane-bound molecules suffers from a number of limitations leading to inaccurate estimation of diffusion parameters. To overcome such problems and with the overall goal of addressing membrane heterogeneities, we performed z-scan FCS (zFCS) of the serotonin_1A receptor. We analyzed the results according to FCS diffusion laws that provide information on the organization of the diffusing species. Analysis of our results shows that the diffusion coefficients of the receptor and a fluorescently labeled phospholipid are similar when probed at length scales ∼210 nm. We discuss the significance of the spatiotemporal evolution of dynamics of membrane-bound molecules in the overall context of membrane domains and heterogeneity. Importantly, our results show that the serotonin_1A receptor exhibits confinement in cell membranes, possibly due to interaction with the actin cytoskeleton. Surprisingly, depletion of membrane cholesterol appears to reduce receptor confinement in a manner similar to that observed in the case of cytoskeletal destabilization, implying possible changes in the actin cytoskeleton induced upon cholesterol depletion. These results constitute the first report on G-protein-coupled receptor dynamics utilizing a combination of zFCS and the FCS diffusion laws, and present a convenient approach to explore cell membrane heterogeneity at the submicron level.     

Activation of Calcium Signaling in Isolated Rat Hepatocytes Is Accompanied by Shape Changes of Microvilli

Preceding studies using the hamster insulinoma cell line, HIT, and isolated rat hepatocytes have shown that two essential components of the Ca²⁺signaling pathway, the ATP-dependent Ca²⁺store and the store-coupled Ca²⁺influx pathway, are both located in microvilli covering the surface of these cells. Microvilli-derived vesicles from both cell types exhibited anion and cation pathways which could be inhibited by anion and cation channel-specific inhibitors. These findings suggested that the microvillar tip compartment forms a space which is freely accessible for external Ca²⁺, ATP, and IP₃. The entry of Ca²⁺into the cytoplasm, however, is largely restricted by the microvillar core structure, the dense bundle of actin microfilaments acting as a diffusion barrier between the microvillar tip compartment and the cell body. Moreover, evidence has been presented that F-actin may function as ATP-dependent and IP₃-sensitive Ca²⁺store that can be emptied by profilin-induced depolymerization or reorganization [K. Lange and U. Brandt (1996)FEBS Lett.395, 137–142]. Here we demonstrate the tight connection between microvillar shape changes and the activation of the Ca²⁺signaling system in isolated rat hepatocytes. Using a combination of scanning electron microscopy (SEM) and fura-2 fluorescence technique, we confirmed a consequence of the “diffusion barrier” concept of Ca²⁺signaling: Irrespective of the type of the applied stimulus, activation of the Ca²⁺influx pathway is accompanied by changes in the structural organization of microvilli indicative of the loss of their diffusion barrier function. We further show that the cell surfaces of unstimulated hepatocytes isolated by either the collagenase or the EDTA perfusion technique are densely covered with microvilli predominantly of a short and slender type. Beside this rather uniformly shaped type of microvilli, a number of dilated surface protrusions were observed. Under these conditions the cells displayed the well known rather high basal [Ca²⁺]_iof 200–250 nMas repeatedly demonstrated for freshly isolated hepatocytes. However, addition of the serine protease inhibitor, phenylmethanesulfonyl fluoride (PMSF), to the cell suspension immediately after its preparation reduced the basal cytoplasmic Ca²⁺level to about 100 nM.Concomitantly, dilated surface protrusions disappeared, and cell surfaces exclusively displayed short, slender microvilli. Activation of the Ca²⁺signaling pathway by vasopressin, as well as by the IP₃-independent acting Ca²⁺store inhibitor, thapsigargin, was accompanied by a conspicuous shortening and dilation of microvilli following the same time courses as the respective increases of [Ca²⁺]_iinduced by the effectors. Furthermore, the abundance of the large form of surface protrusions on isolated hepatocytes positively correlated with the size of a cellular Ca²⁺/Fura-2 compartment which is rapidly depleted from Ca²⁺by extracellular EGTA. These findings support the postulated localization of the store-coupled Ca²⁺influx pathway in microvilli of HIT cells also for hepatocytes and are in accord with the notion of a cytoskeletal diffusion barrier regulating the flux of external Ca²⁺via the microvillar tip region in the cytoplasm.     

Alteration of Synaptosomal Plasma Membrane Cholesterol Content: Membrane Physical Properties and Cation Transport Proteins

The level of nerve membrane cholesterol was altered by in vitro incubation of rat brain synaptosomal plasma membrane with liposomes having varying cholesterol contents. The normal plasma membrane cholesterol/phospholipid ratio of 0.3-0.4 (mol/mol) could be decreased by about one-half or increased more than 100%. Fluorescence polarization measurements were made using the probe 1,6-diphenyl-1,3,5-hexatriene. At temperatures below 35 percent C, lowering membrane cholesterol led to increased apparent microviscosity, while raising cholesterol content produced little change. However, at 45 percent C a continuous direct relationship existed between experimental membrane cholesterol/phospholipid ratio (ranging from 0.18 to 0.73) and apparent microviscosity. Under standard liposome-synaptosomal plasma membrane exchange conditions, 80% of the initial specific [(3)H]saxitoxin binding activity to the voltage-dependent sodium channel and at least 95% of the (Mg2+,K+)-p-nitrophenylphosphatase activity were preserved. Our results indicate that neither the characteristics of toxin binding nor the kinetics of this enzyme activity is dependent upon membrane cholesterol content.     

Anti-Arrhythmic Effect of Verapamil Is Accompanied by Preservation of Cx43 Protein in Rat Heart

The present study was to test the hypothesis that anti-arrhythmic properties of verapamil may be accompanied by preserving connexin43 (Cx43) protein via calcium influx inhibition. In an in vivo study, myocardial ischemic arrhythmia was induced by occlusion of the left anterior descending (LAD) coronary artery for 45 min in Sprague-Dawley rats. Verapamil, a calcium channel antagonist, was injected i.v. into a femoral vein prior to ischemia. Effects of verapamil on arrhythmias induced by Bay K8644 (a calcium channel agonist) were also determined. In an ex vivo study, the isolated heart underwent an initial 10 min of baseline normal perfusion and was subjected to high calcium perfusion in the absence or presence of verapamil. Cardiac arrhythmia was measured by electrocardiogram (ECG) and Cx43 protein was determined by immunohistochemistry and western blotting. Administration of verapamil prior to myocardial ischemia significantly reduced the incidence of ventricular arrhythmias and total arrhythmia scores, with the reductions in heat rate, mean arterial pressure and left ventricular systolic pressure. Verapamil also inhibited arrhythmias induced by Bay K8644 and high calcium perfusion. Effect of verapamil on ischemic arrhythmia scores was abolished by heptanol, a Cx43 protein uncoupler and Gap 26, a Cx43 channels inhibitor. Immunohistochemistry data showed that ischemia-induced redistribution and reduced immunostaining of Cx43 were prevented by verapamil. In addition, diminished expression of Cx43 protein determined by western blotting was observed following myocardial ischemia in vivo or following high calcium perfusion ex vivo and was preserved after verapamil administration. Our data suggest that verapamil may confer an anti-arrhythmic effect via calcium influx inhibition, inhibition of oxygen consumption and accompanied by preservation of Cx43 protein.     

Isolation and Characterization of the Plasma Membrane by Two-Phase Partitioning from the Alkalophilic Cyanobacterium Spirulina platensis

Partition in aqueous dextran-polyethylene glycol two-phase systemwas used to isolate the plasma membranes from the alkalophiliccyanobacterium Spirulina platensis. The upper phase containeda colorless membranes obtained in relatively short time, 3–4h. This fraction had a different protein profile than that ofthe thylakoid fraction obtained in the lower phase. It did notcontain cytochrome c-oxidase activity, but retained characteristicMg²⁺-ATPase activity that is sensitive to vanadate, stimulatedby K⁺, and has a pH optimum near 8.5. These data support ourassumption that the upper phase of the gradient consist of theplasma membrane of S. platensis. (Received November 25, 1993; Accepted April 12, 1994)