Membrane Biology

BACKGROUND:
In spite of its indusrial usefulness and varied daily uses, lead (Pb) pollution is a widespread ecological problem that faces the humans in the 21th century. Pb was found to produces a wide range of toxic effects including neurotoxicity especially to the developing and young offspring. Recently, the utilization of herbal plants has received a significant attention where there has been rising awareness in their therapeutic use; among these is the garlic.
AIM OF THE WORK:
In light of the above, the current study is designed experimentally in female pregnant rats in order to investigate the beneficial role of garlic extract in the protection from the maternal and fetal cerebellar damage that produced by administration of different doses of Pb during pregnancy.
MATERIALS AND METHODS:
Positively pregnant female rats were divided into five groups; one control group, two Pb-treated groups (exposed to 160 and 320 mg/kg b.wt. of Pb, respectively) and two groups treated with both Pb and garlic (exposed to Pb as previous groups together with 250 mg/ kg b.wt. /day of garlic extract). Treatments started from day 1 till day 20 of pregnancy, where the mother rats of different experimental groups were sacrified to obtain the fetuses. Pb level in the maternal nd fetal blood and cerebellum was estimated by spectrophotometry. Specimens of the cerebellum of different mother and fetal groups were processed to histological and immunohistochemical staining for microscopic examination.
RESULTS:
The results showed that administration of Pb to pregnant rats resulted in a dose-dependent toxicity for both mothers and fetuses in the form of decrease of maternal weight gain, placental and fetal weights, brain weight and diminished fetal growth parameters, which were prominent in rat's group treated with larger dose of Pb. In Pb-treated rats, Pb level in blood and cerebellum was high when compared to the control. The histopathological examination of the cerebellum of treated dams and fetuses showed marked alterations mainly in the form of Purkinje cell degeneration and lack of deveopmet of fetal cerebellum. Co-treatment of garlic extract along with Pb resulted in a significant decrease in Pb levels as compared with those treated with Pb alone with improvement of the histopathological changes.
CONCLUSIONS:
This study was useful in evaluating the hazardous effects of uncontrolled use of Pb in general and in assessing the developmental and neurotoxicity of fetuses due to exposure during pregnancy in particular. Co-administration of garlic has beneficial effects in amelioration of Pb-induced neurotoxicity and reversing the histopathological changes of the cerebellum of mother rats and fetuses.
KEYWORDS:
garlic; glial fibrillary acidic protein; lead; purkinje cells

Triton X-100 extracted ciliary membrane protein from isolated cilia, prepared from the protozoon Tetrahymena thermophila, were fractionated by affinity chromatography on columns with covalently bound fibroblast growth factor (FGF), insulin, or concanavalin A (ConA), respectively. The eluted proteins were further analyzed by electrophoresis on sodium dodecyl sulfate polyacrylamide gels, isoelectric focusing, and by immunoblotting techniques using antibodies against the FGF receptor, platetelet derived growth factor (PDGF) receptor ␣-subunit, and insulin receptor ␤-subunit. The particular antibodies were chosen because the peptides PDGF, FGF, insulin, and ConA are chemoattractants in this organism and corresponding binding (receptor) proteins could be expected to be identified.

The Fluid-Mosaic Membrane Model of cell
membrane structure was based on thermodynamic
principals and the available data on component
lateral mobility within the membrane plane [Singer
SJ, Nicolson GL. The Fluid Mosaic Model of the
structure of cell membranes. Science 1972; 175:
720-731]. After more than forty years the model
remains relevant for describing the basic nano-scale
structures of a variety of biological membranes.
More recent information, however, has shown the
importance of specialized membrane domains, such
as lipid rafts and protein complexes, in describing
the macrostructure and dynamics of biological
membranes. In addition, membrane-associated
cytoskeletal structures and extracellular matrix also
play roles in limiting the mobility and range of
motion of membrane components and add new
layers of complexity
and hierarchy to the
original model. An
updated Fluid-Mosaic
Membrane Model is
described, where more
emphasis has been
placed on the mosaic nature of cellular membranes
where protein and lipid components are more
crowded and limited in their movements in the
membrane plane by lipid-lipid, protein-protein and
lipid-protein interactions as well as cell-matrix, cellcell
and cytoskeletal interactions. These interactions
are important in restraining membrane components
and maintaining the unique mosaic organization of
cell membranes into functional, dynamic domains.

Microfluidics has facilitated immunological studies by enhancing speed, efficiency and sensitivity of current analysis methods. It offers miniaturization of current laboratory equipment, and enables analysis of clinical samples without the need for sophisticated infrastructure. More importantly, microfluidics offers unique capabilities; including conducting multiple serial or parallel tasks as well as providing complex and precisely controlled environmental conditions that are not achievable using conventional laboratory equipment. Microfluidics is a promising technology for fundamental and applied immunological studies, allowing generation of high throughput, robust and portable platforms, opening a new area of automation in immunology.

The key to the keys to immortality and eternal youth lies in the correct answer to the main question: How to naively discover new essential – but still hidden – features required for properly training novel adaptive supervised machine learning algorithms (SMLA)

Within the philosophy of biology, recently promising steps have been made towards a biologically grounded concept of agency. Agency is described as bio-agency: the intrinsically normative adaptive behaviour of human and non-human organisms, arising from their biological autonomy.
My paper assesses the bio-agency approach by examining criticism recently directed by its proponents against the project of embodied robotics. Defenders of the bio-agency approach have claimed that embodied robots do not, and for fundamental reasons cannot, qualify as artificial agents because they do not fully realise biological autonomy. More particularly, it has been claimed that they fail to be agents because agency essentially requires metabolism.
I shall argue that this criticism, while being valuable in bringing to the fore important differences between bio-agents and existing embodied robots, nevertheless is too strong. It relies on inferences from agency-as-we-know-it to agency-as-it-could-be which are justified neither empirically nor conceptually.

An excess of intracellular free Cholesterol (Chol) is cytotoxic, and its homeostasis is crucial for cell viability. Apolipoprotein A-I (apoA-I) is a highly efficient Chol acceptor as it activates complex cellular pathways that tend to mobilize and export Chol from cellular depots. Here we hypothesize that membrane composition and/or organization is strongly involved in Chol homeostasis. To test this hypothesis, we constructed a cell line over expressing Stearoyl CoA desaturase (SCD-cells), which modifies plasma membrane (PM) composition by the enrichment of monounsaturated fatty,acids and determined this effect on membrane properties, cell viability and cholesterol homeostasis. PM in SCD-cells has a higher phospholipids/sphingomyelin ratio and is slightly enriched in Chol. These cells showed an increase in the cholesteryl esters/free Chol ratio, they were more resistant to Chol toxicity and in addition, they exported more caveolin than Control cells. The data suggest that cell functionality is preserved by regulating membrane fluidity and Chol exportation and storage.

Intense nanosecond pulsed electric fields (nsPEFs) have been shown to induce, on intracellular structures, interesting effects dependent on electrical exposure conditions (pulse length and amplitude, repetition frequency and number of pulses), which are known in the literature as “bioelectrical effects” (Schoenbach et al., IEEE Trans Plasma Sci 30:293–300, 2002). In particular, pulses with a shorter width than the plasma membrane charging time constant (about 100 ns for mammalian cells) can penetrate the cell and trigger effects such as permeabilization of intracellular membranes, release of Ca2+ and apoptosis induction. Moreover, the observed effects have led to exploration of medical applications, like the treatment of melanoma tumors (Nuccitelli et al., Biochem Biophys Res Commun 343:351–360, 2006). Pulsed electric fields allowing such effects usually range from several tens to a few hundred nanoseconds in duration and from a few to several tens of megavolts per meter in amplitude (Schoenbach et al., IEEE Trans Diel Elec Insul 14:1088–1109, 2007); however, the biological effects of subnanosecond pulses have been also investigated (Schoenbach et al., IEEE Trans Plasma Sci 36:414–422, 2008). The use of such a large variety of pulse parameters suggests that highly flexible pulse-generating systems, able to deliver wide ranges of pulse durations and amplitudes, are strongly required in order to explore effects and applications related to different exposure conditions. The Blumlein pulse-forming network is an often-employed circuit topology for the generation of high-voltage electric pulses with fixed pulse duration. An innovative modification to the Blumlein circuit has been recently devised which allows generation of pulses with variable amplitude, duration and polarity. Two different modified Blumlein pulse-generating systems are presented in this article, the first based on a coaxial cable configuration, matching microscopic slides as a pulse-delivery system, and the other based on microstrip transmission lines and designed to match cuvettes for the exposure of cell suspensions.

Lens Major Intrinsic Protein (MIP) is a member of a family of membrane transport proteins including the Aquaporins and bacterial glycerol transporters. When expressed in Xenopus oocytes, MIP increased both glycerol permeability and the activity of glycerol kinase. Glycerol permeability (p Gly ) was 2.3 ± 0.23 × 10 −6 cm sec −1 with MIP vs. 0.92 ± 0.086 × 10 −6 cm sec −1 in control oocytes. The p Gly of MIP was independent of concentration from 5 × 10 −5 to 5 × 10 −2 M, had a low temperature dependence, and was inhibited approximately 90%, 80% and 50% by 1.0 mM Hg ++ , 0.2 mM DIDS (diisothiocyanodisulfonic stilbene), and 0.1 mM Cu ++ , respectively. MIP-enhanced glycerol phosphorylation, resulting in increased incorporation of glycerol into lipids. This could arise from an increase in the total activity of glycerol kinase, or from an increase in its affinity for glycerol. Based on methods we present to distinguish these mechanisms, MIP increased the maximum rate of phosphorylation by glycerol kinase (0.12 ± 0.03 vs. 0.06 ± 0.01 pmol min −1 cell −1 ) without changing the binding of glycerol to the kinase (K M ∼ 10 M).

This paper investigates one of the great achievements of 20th-century cell biology: determining the structure of the cell membrane. This case differs in important ways from the better-known case of the identification of the DNA double helix as the carrier of genetic information, especially regarding the evaluation of potential evidence in light of prior theoretical commitments. Whereas it has been argued that adherence to a structural hypothesis enabled Watson and Crick to ignore a surplus of (potentially confusing) empirical findings, similar adherence to an elegant and universal structural hypothesis, we argue, unduly shielded the so-called 'unit-membrane' model from legitimate challenges on the basis of known phenomena.

Sertoli cells play a key role in the establishment of an adequate luminal environment in the seminiferous tubules of the male reproductive tract. Secretion of the seminiferous tubular fluid (STF) is vital for the normal occurrence of spermatogenesis and for providing a means of transport to the developing spermatozoa. However, several studies on this subject have not completely clarified the origin and composition of this fluid. Electrolyte and water are central components of STF. Sertoli cells secrete an iso-osmotic fluid with a higher content of K+ than the blood and express various membrane and water transporters (Na+/K+-ATPase; Ca2+-ATPase; V-type ATPase; Cl− channels; CFTR Cl− channels; K+ channels; L-, T- and N-type Ca2+ channels; Na+/H+ exchangers; Na+-driven HCO3−/Cl− exchangers (NDCBEs); Na+/HCO3− cotransporters (NBCes); Na+–K+–2Cl− cotransporter; Na+/Ca2+ exchanger; and aquaporins 0 and 8) involved in cellular and secretory functions. Studies with knockout mice for some of these transporters showed tubular fluid accumulation and associated infertility, revealing the relevance of these processes for the normal occurrence of spermatogenesis. Nevertheless, the role of the several membrane transporters in the establishment of STF electrolyte composition needs to be further elucidated. This review summarizes the available data on the ionic composition of STF and on the Sertoli cell membrane mechanisms responsible for ion and water movement. Deepening the knowledge on the mechanisms involved in the secretion, composition and regulation of SFT is essential and will be a major step in understanding the infertility associated with some pathological conditions.

We have previously reported that rat parotid gland basolateral plasma membrane vesicles (BLMV) have a relatively high affinity Ca 2 § transport pathway and an unsaturable Ca 2+ flux component (Lockwich et al., 1994. J. Membrane Biol. 141:289-296). In this study, we have solubilized BLMV with octylglucoside (1.5%) and have reconstituted the solubilized proteins into proteoliposomes (PrL) composed of E. coli bulk phospholipids, by using a detergent dilution method. PrL exhibited 3-5-fold higher 45Ca2 § influx than control liposomes (without protein). Ca 2 § uptake into PrL was dependent on the [protein] in PrL and steady state [Ca >] in PrL was in equilibrium with external [Ca2+]. These data demonstrate that a passive, protein-mediated Ca 2 § transport has been reconstituted from BLMV into PrL. 45Ca2+ influx into liposomes did not saturate with increasing [Ca 2+] in the assay medium. In contrast, PrL displayed saturable 45Ca2+ influx and exhibited a single Ca 2+ flux component with an apparent Kca = 242 + 50.9 gM and Vma x = 13.5 + 1.14 nmoles Ca2+/mg protein/ minute. The Kc~ of Ca2+-transport in PrL was similar to that of the high affinity Ca a+ influx component in BLMV while the gma x was about 4-fold higher. The unsaturable Ca 2 § flux component was not detected in PrL. 45Ca2+ influx in PrL was inhibited by divalent cations in the order of efficacy, Zn 2+ > Mn 2+ > Co 2 § = Ni 2 § and appeared to be more sensitive to lower concentrations of Zn 2 § than in BLMV. Consistent with our observations with BLMV, the carboxyl group reagent N,N'-dicyclohexylcarbodiimide (DCCD) inhibited the reconstituted * Present address: Ca 2+ transport in PrL. Importantly, in both BLMV and PrL, DCCD induced a 40-50% decrease in Vm~ ~ of Ca 2+ transport without an alteration in Kca. These data strongly suggest that the high affinity, passive Ca 2+ transport pathway present in BLMV has been functionally reconstituted into PrL. We suggest that this approach provides a useful experimental system towards isolation of the protein(s) involved in mediating Ca 2+ influx in the rat parotid gland basolateral plasma membrane.

Voltage-dependent ion channels are a difficult class of proteins to approach biochemically. Many such channels are present at low density in relevant tissues and exist as multiple subtypes that can be distinguished electrophysiologically. In particular, K channels appear to be a diverse family of proteins characterized by many different conductance properties, gating behaviors and regulatory phenomena. Fortunately, specific peptide toxins for K channels are present in the venoms of insects, scorpions, snakes and possibly other species. The available sequences of these peptides define several different families of toxins. Electrophysiological and radioligand binding studies suggest that these toxins can be used to distinguish subclasses of K channels that share similar toxin binding sites. The growing databank of sequence homologies for both toxins and channels is, in essence, a codebook for identifying common elements of structure and function. The continuing development of toxins as biochemical probes should help to uncover the molecular basis and physiological significance of K-channel diversity.

The interplay between the amyloid-β (Aβ) peptide and cellular membranes have been proposed as an important mechanism for toxicity in Alzheimer’s disease (AD). Membrane environments appear to influence Aβ aggregation and may stabilize intermediate Aβ oligomeric states that are considered to be neurotoxic. One important role for molecular biophysics within the field of Aβ studies is to characterize the structure and dynamics of the Aβ peptide in various states, as well as the kinetics of transfer between these states. Because biological cell membranes are very complex, simplified membrane models are needed to facilitate studies of Aβ and other amyloid proteins in lipid environments. In this review, we examine different membrane-mimetic systems available for molecular studies of Aβ. An introduction to each system is given, and examples of important findings are presented for each system. The benefits and drawbacks of each system are discussed from methodical and biological perspectives.

An in-depth understanding of the mechanisms underlying regulatory volume behavior in corneal epithelial cells has been in part hampered by the lack of adequate methodology for characterizing this phenomenon. Accordingly, we developed a novel approach to characterize time-dependent changes in relative cell volume induced by anisosmotic challenges in calcein-loaded SV40-immortalized human corneal epithelial (HCE) cells with a fluorescence microplate analyzer. During a hypertonic challenge, cells shrank rapidly, followed by a temperature-dependent regulatory volume increase (RVI), τc = 19 min. In contrast, a hypotonic challenge induced a rapid (τc = 2.5 min) regulatory volume decrease (RVD). Temperature decline from 37 to 24°C reduced RVI by 59%, but did not affect RVD. Bumetanide (50 μM), ouabain (1 mM), DIDS (1 mM), EIPA (100 μM), or Na+-free solution reduced the RVI by 60, 61, 39, 32, and 69%, respectively. K+, Cl− channel and K+-Cl− cotransporter (KCC) inhibition obtained with either 4-AP (1 mM), DIDS (1 mM), DIOA (100 μM), high K+ (20 mM) or Cl−-free solution, suppressed RVD by 42, 47, 34, 52 and 58%, respectively. KCC activity also affects steady-state cell volume, since its inhibition or stimulation induced relative volume alterations under isotonic conditions. Taken together, K+ and Cl− channels in parallel with KCC activity are important mediators of RVD, whereas RVI is temperature-dependent and is essentially mediated by the Na+-K+-2Cl− cotransporter (Na+-K+-2Cl−) and the Na+-K+ pump. Inhibition of K+ and Cl− channels and KCC but not Na+-K+-2Cl− affect steady-state cell volume under isotonic conditions. This is the first report that KCC activity is required for HCE cell volume regulation and maintenance of steady-state cell volume.

Pandinus imperator scorpion toxins Pi2 and Pi3 differ only by a single amino acid residue (neutral Pro7 in Pi2 vs. acidic Glu7 in Pi3). The binding kinetics of these toxins to human Kv1.3 showed that the decreased on rate (k ON ‫ס‬ 2.18 × 10 8 M −1 sec −1 for Pi2 and 1.28 × 10 7 M −1 sec −1 for Pi3) was almost entirely responsible for the increased dissociation constant (K d ) of Pi3 (K d ‫ס‬ 795 pM) as compared to Pi2 (K d ‫ס‬ 44 pM). The ionic strength dependence of the association rates was exactly the same for the two toxins indicating that through-space electrostatic interactions can not account for the different on rates. Results were further analyzed on the basis of the three-dimensional structural models of the toxins. A 3D structure of Pi3 was generated from the NMR spectroscopy coordinates of Pi2 by computer modeling. The Pi3 model resulted in a salt bridge between Glu7 and Lys24 in Pi3. Based on this finding our interpretation of the reduced on rate of Pi3 is that the intramolecular salt bridge reduces the local positive electrostatic potential around Lys24 resulting in decreased short-range electrostatic interactions during the binding step. To support our finding, we constructed a 3D model of the Ser-10-Asp Charybdotoxin mutant displaying distinctly reduced affinity for Shaker channels. The mutant Charybdotoxin structure also displayed a salt bridge between residues Asp10 and Lys27 equivalent to the one between Glu7 and Lys24 in Pi3.

Two novel peptides were purified from the venom of the scorpion Pandinus imperator, and were named Pi2 and Pi3. Their complete primary structures were determined and their blocking effects on Shaker B K + channels were studied. Both peptides contain 35 amino acids residues, compacted by three disulfide bridges, and reversibly block the Shaker B K + channels. They have only one amino acid changed in their sequence, at position 7 (a proline for a glutamic acid). Whereas peptide Pi2, containing the Pro7, binds the Shaker B K + channels with a K d of 8.2 nM, peptide Pi3 containing the Glu7 residue has a much lower affinity of 140 nM. Both peptides are capable of displacing the binding of 125 I-noxiustoxin to brain synaptosome membranes. Since these two novel peptides are about 50% identical to noxiustoxin, the present results support previous data published by our group showing that the amino-terminal region of noxiustoxin, and also the amino-terminal sequence of the newly purified homologues: Pi2, and Pi3, are important for the recognition of potassium channels.

We have identified a protein of 110 kDa in the renal epithelial cell line NBL-1, which is induced on incubation of the cells in an amino-acid-free medium. The protein was purified on conA-Sepharose and subjected to N-terminal sequencing. The sequence obtained, VDRINFKT, does not correspond to any protein in the databases. Antipeptide antibodies made to this sequence recognised a single protein of 110 kDa in whole cell membranes and in a conconavalin A protein extract. Using the antibody on

The use of plasmid DNA (pDNA) as a pharmaceutical tool has increased since it represents a safer vector for gene transfer compared to viral vectors. Different pDNA extraction methods have been described; among them is alkaline lysis, currently the most commonly used. Although alkaline lysis represents an established method for isolation of pDNA, some drawbacks are recognized, such as entrapment of pDNA in cell debris, leading to lower pDNA recovery; the time-consuming process; and increase of the volume due to the buffers used, all leading to increased cost of production. We compared the concentration of extracted pDNA when two methods for extracting pDNA from Escherichia coli were used: alkaline lysis and a method based on membrane electroporation, electroextraction. At the same time, we also studied the effect of different pulse protocols on bacterial inactivation. The concentration of pDNA was assayed with anion exchange chromatography. When alkaline lysis was used, two incubations of lysis time (5 and 10 min) were compared in terms of the amount of isolated pDNA. We did not observe any difference in pDNA concentration regardless of incubation time used. In electroextraction, different pulse protocols were used in order to exceed the pDNA concentration obtained by alkaline lysis. We show that electroextraction gives a higher concentration of extracted pDNA than alkaline lysis, suggesting the use of electroporation as a potentially superior method for extracting pDNA from E. coli. In addition, electroextraction represents a quicker alternative to alkaline lysis for extracting pDNA.

In this present study, the root extracts of Withania somnifera were subjected to a comparative evaluation of the membrane stabilization and thrombolytic activity on human blood sample. The thrombolytic and membrane stabilizing activities were assessed by using human erythrocyte and the results were compared with standard streptokinase (SK) and standard anti-inflammatory drug, acetyl salicylic acid (ASA), respectively. The Methanol extract showed the highest thrombolytic activity (68.14%). On the other hand hypotonic solution induced haemolysis, at a concentration of 1.0 mg/ mL, the methanol extract inhibited 71.90% haemolysis of RBCs as compared to 73.56% produced by acetyl salicylic acid (0.10 mg/mL). And in case of heat induced condition different organic soluble materials of Withania somnifera demonstrated 54.03%, 53.13% and 38.54% inhibition of hemolysis of RBCs, respectively whereas ASA inhibited 48.10%.

Several effects of the endogenous opioid peptide dynorphin A (Dyn A) are not mediated through the opioid receptors. These effects are generally excitatory, and result in cell loss and induction of chronic pain and paralysis. The mechanism(s) is not well defined but may involve formation of pores in cellular membranes. In the 17-amino acid peptide Dyn A we have recently identified L5S, R6W, and R9C mutations that cause the dominantly inherited neurodegenerative disorder Spinocerebellar ataxia type 23. To gain further insight into non-opioid neurodegenerative mechanism(s), we studied the perturbation effects on lipid bilayers of wild type Dyn A and its mutants in large unilamellar phospholipid vesicles encapsulating the fluorescent dye calcein. The peptides were found to induce calcein leakage from uncharged and negatively charged vesicles to different degrees, thus reflecting different membrane perturbation effects. The mutant Dyn A R6W was the most potent in producing leakage with negatively charged vesicles whereas Dyn A L5S was virtually inactive. The overall correlation between membrane perturbation and neurotoxic response [3] suggests that pathogenic Dyn A actions may be mediated through transient pore formation in lipid domains of the plasma membrane.

The sodium bicarbonate cotransporter (NBC1) is essential for bicarbonate transport across plasma membranes in epithelial and nonepithelial cells. The direction of the NaHCO 3 movement in secretory epithelia is opposite to that in reabsorptive epithelia. In secretory epithelia (such as pancreatic duct cells) NBC is responsible for the transport of bicarbonate from blood to the cell for eventual secretion at the apical membrane. In reabsorptive epithelia (such as kidney proximal tubule cells) NBC is responsible for the reabsorption of bicarbonate from cell to the blood. In nonepithelial cells this transporter is mainly involved with cell pH regulation. Recent molecular cloning experiments have identified the existence of four NBC isoforms (NBC1, 2, 3 and 4) and two NBCrelated proteins AE4 and NCBE (Anion Exchanger 4 and Na-dependent Chloride-Bicarbonate Exchanger). All but AE4 are presumed to mediate the cotransport of Na + and HCO 3 − under normal conditions and may be functionally altered in certain pathologic states. NBC1 shows a limited tissue expression pattern, is electrogenic and plays an important role in bicarbonate reabsorption in kidney proximal tubule. In addition to the kidney, NBC1 is expressed in pancreatic duct cells, is activated by cystic fibrosis transmembrane conductance regulator (CFTR) and plays an important role in HCO 3 − secretion. NBC2 and NBC3 have a wider tissue distribution than NBC1, are electroneutral, and are involved with cell pH regulation. The characterization of NBC4 is incomplete. The NBC-related protein called NCBE mediates Nadependent, Cl − /Bicarbonate Exchange. The purpose of this review is to summarize recent advances on the cloning of NBC isoforms and related proteins and their role and regulation in physiologic and pathologic states.

Langerhans are thought to be involved in death of the insulinproducing islet ␤ cells in type 2 diabetes mellitus. It has been suggested that the mechanism of this ␤ cell death involves membrane disruption by human islet amyloid polypeptide (hIAPP), the major constituent of islet amyloid. However, the molecular mechanism of hIAPP-induced membrane disruption is not known. Here, we propose a hypothesis that growth of hIAPP fibrils at the membrane causes membrane damage. We studied the kinetics of hIAPP-induced membrane damage in relation to hIAPP fibril growth and found that the kinetic profile of hIAPP-induced membrane damage is characterized by a lag phase and a sigmoidal transition, which matches the kinetic profile of hIAPP fibril growth. The observation that seeding accelerates membrane damage supports the hypothesis. In addition, variables that are well known to affect hIAPP fibril formation, i.e., the presence of a fibril formation inhibitor, hIAPP concentration, and lipid composition, were found to have the same effect on hIAPP-induced membrane damage. Furthermore, electron microscopy analysis showed that hIAPP fibrils line the surface of distorted phospholipid vesicles, in agreement with the notion that hIAPP fibril growth at the membrane and membrane damage are physically connected. Together, these observations point toward a mechanism in which growth of hIAPP fibrils, rather than a particular hIAPP species, is responsible for the observed membrane damage. This hypothesis provides an additional mechanism next to the previously proposed role of oligomers as the main cytotoxic species of amyloidogenic proteins.

In vitro and in vivo studies support the involvement of connexin 43-based cell-cell channels and hemichannels in cell death propagation induced by ischemia-reperfusion. In this context, open connexin hemichannels in the plasma membrane have been proposed to act as accelerators of cell death. Progress on the mechanisms underlying the cell permeabilization induced by ischemia-reperfusion reveals the involvement of several factors leading to an augmented open probability and increased number of hemichannels on the cell surface.

Earlier studies have suggested a role for Ca 2+ in regulatory volume decrease (RVD) in response to hypotonic stress through the activation of Ca 2+ -dependent ion channels . The involvement of Ca 2+ in regulating cell volume in rat lacrimal acinar cells was therefore examined using a video-imaging technique to measure cell volume. The trivalent cation Gd 3+ inhibited RVD, suggesting that Ca 2+ entry is important and may be via stretch-activated cation channels. However, Fura-2 loaded cells did not show an increase in [Ca 2+ ] i during exposure to hypotonic solutions. The absence of any changes in [Ca 2+ ] i resulted from the buffering of cytosolic Ca 2+ by Fura-2 during hypotonic shock and therefore inhibition of RVD. The intracellular Ca 2+ chelator, BAPTA, also inhibited the RVD response to hypotonic shock. An increase in [Ca 2+ ] i induced by either acetylcholine or ionomycin, was found to decrease cell volume under isotonic conditions in lacrimal acinar cells. Cell shrinkage was inhibited by tetraethylammonium ion, an inhibitor of Ca 2+activated K + channels. On the basis of the presented data, we suggest an involvement of intracellular Ca 2+ in controlling cell volume in lacrimal acinar cells.

By means of cryoelectron transmission microscopy (cryo-TEM), we were able to demonstrate the formation of ternary complexes (TC): DNA–phosphatidylcholine liposome–divalent metal cations. Addition of Ba2+ to TC led to visualization of DNA compacting on the liposome surface. Staining the TC by Tb3+ cations revealed the changed secondary structure of DNA located between fused liposomes. Cryo-TEM and liposome turbidity data were analyzed during TC formation. Liposome aggregation and the liposome fusion induced by DNA in TC were observed. Because TC displayed the property of DNA cationic liposome complexes as well as their own unique properties, we were able to consider cationic lipoplexes as a particular case of TC. The involvement of TC and direct DNA–lipid interactions in the formation nuclear pore complexes were assumed.

Using 237 all-atom double bilayer simulations, we examined the thermodynamic and structural changes that occur as a phosphatidylcholine lipid bilayer stack is dehydrated. The simulated system represents a micropatch of lipid multilayer systems that are studied experimentally using surface force apparatus, atomic force microscopy and osmotic pressure studies. In these experiments, the hydration level of the system is varied, changing the separation between the bilayers, in order to understand the forces that the bilayers feel as they are brought together. These studies have found a curious, strongly repulsive force when the bilayers are very close to each other, which has been termed the ''hydration force,'' though the origins of this force are not clearly understood. We computationally reproduce this repulsive, relatively free energy change as bilayers come together and make qualitative conclusions as to the enthalpic and entropic origins of the free energy change. This analysis is supported by data showing structural changes in the waters, lipids and salts that have also been seen in experimental work. Increases in solvent ordering as the bilayers are dehydrated are found to be essential in causing the repulsion as the bilayers come together.

Quantitative structures are obtained at 30°C for the fully hydrated fluid phases of palmitoyloleoylphosphatidylcholine (POPC), with a double bond on the sn-2 hydrocarbon chain, and for dierucoylphosphatidylcholine (di22:1PC), with a double bond on each hydrocarbon chain. The form factors F(q z ) for both lipids are obtained using a combination of three methods. (1) Volumetric measurements provide F(0). (2) X-ray scattering from extruded unilamellar vesicles provides jF(q z )j for low q z . (3) Diffuse X-ray scattering from oriented stacks of bilayers provides jF(q z )j for high q z . Also, data using method (2) are added to our recent data for dioleoylphosphatidylcholine (DOPC) using methods (1) and ; the new DOPC data agree very well with the recent data and with (4) our older data obtained using a liquid crystallographic X-ray method. We used hybrid electron density models to obtain structural results from these form factors. The result for area per lipid (A) for DOPC 72.4 ± 0.5 Å 2 agrees well with our earlier publications, and we find A = 69.3 ± 0.5 Å 2 for di22:1PC and A = 68.3 ± 1.5 Å 2 for POPC. We obtain the values for five different average thicknesses: hydrophobic, steric, head-head, phosphate-phosphate and Luzzati. Comparison of the results for these three lipids and for our recent dimyristoylphosphatidylcholine (DMPC) determination provides quantitative measures of the effect of unsaturation on bilayer structure. Our results suggest that lipids with one monounsaturated chain have quantitative bilayer structures closer to lipids with two monounsaturated chains than to lipids with two completely saturated chains.

The electrochemical properties of a widely accepted six-step reaction scheme for the Na+, K+-ATPase have been studied by computer simulation. Rate coefficients were chosen to fit the nonvectorial biochemical data for the isolated enzyme and a current-voltage (I-V) relation consistent with physiological observations was obtained with voltage dependence restricted to one (but not both) of the two translocational steps. The vectorial properties resulting from these choices were consistent with physiological activation of the electrogenic sodium pump by intracellular and extracellular sodium (Na+) and potassium (K+) ions. The model exhibited K+/K+ exchange but little Na+/Na+ exchange unless the energy available from the splitting of adenosine triphosphate (ATP) was reduced, mimicking the behavior seen in squid giant axon. The vectorial ionic activation curves were voltage dependent, resulting in large shifts in apparent Km's with depolarization. At potentials more negative than the equilibrium or reversal potential transport was greatly diminished unless the free energy of ATP splitting was reduced. While the pump reversal potential is at least 100 mV hyperpolarized relative to the resting potential of most cells, the voltage-dependent distribution of intermediate forms of the enzyme allows the possibility of considerable slope conductance of the pump I-V relation in the physiological range of membrane potentials. Some of the vectorial properties of an electrogenic sodium pump appear to be inescapable consequences of the nonvectorial properties of the isolated enzyme. Future application of this approach should allow rigorous quantitative testing of interpretative ideas concerning the mechanism and stoichiometry of the sodium pump.

Bacterial and archaeal cell envelopes are complex multilayered barriers that serve to protect these microorganisms from their extremely harsh and often hostile environments. Import of exogenous proteins and nanoparticles into cells is important for biotechnological applications in prokaryotes. In this report, we demonstrate that cell-penetrating peptides (CPPs), both bacteriaexpressed nona-arginine peptide (R9) and synthetic R9 (SR9), are able to deliver noncovalently associated proteins or quantum dots into four representative species of prokaryotes: cyanobacteria (Synechocystis sp. PCC 6803), bacteria (Escherichia coli DH5a and Arthrobacter ilicis D-50), and archaea (Thermus aquaticus). Although energydependent endocytosis is generally accepted as a hallmark that distinguishes eukaryotes from prokaryotes, cellular uptake of uncomplexed green fluorescent protein (GFP) by cyanobacteria was mediated by classical endocytosis. Mechanistic studies revealed that macropinocytosis plays a critical and major role in CPP-mediated protein transduction in all four prokaryotes. Membrane damage was not observed when cyanobacterial cells were treated with R9/ GFP complexes, nor was cytotoxicity detected when bacteria or archaea were treated with SR9/QD complexes in the presence of macropinocytic inhibitors. These results indicate that the uptake of protein is not due to a compromise of membrane integrity in cyanobacteria, and that CPP can be an effective and safe carrier for membrane trafficking in prokaryotic cells. Our investigation provides important new insights into the transport of exogenous proteins and nanoparticles across the complex membrane systems of prokaryotes.

Caffeine causes a [Ca 2+ ] i increase in the cortex of Paramecium cells, followed by spillover with considerable attenuation, into central cell regions. From [Ca 2+ ] i rest ∼50 to 80 nM, [Ca 2+ ] i act rises within ഛ3 sec to 500 (trichocyst-free strain tl) or 220 nM (nondischarge strain nd9-28°C) in the cortex. Rapid confocal analysis of wildtype cells (7S) showed only a 2-fold cortical increase within 2 sec, accompanied by trichocyst exocytosis and a central Ca 2+ spread during the subsequent ജ2 sec. Chelation of Ca o 2+ considerably attenuated [Ca 2+ ] i increase. Therefore, caffeine may primarily mobilize cortical Ca 2+ pools, superimposed by Ca 2+ influx and spillover (particularly in tl cells with empty trichocyst docking sites). In nd cells, caffeine caused trichocyst contents to decondense internally (Ca 2+ -dependent stretching, normally occurring only after membrane fusion). With 7S cells this usually occurred only to a small extent, but with increasing frequency as [Ca 2+ ] i signals were reduced by [Ca 2+ ] o chelation. In this case, quenched-flow and ultrathin section or freeze-fracture analysis revealed dispersal of membrane components (without fusion) subsequent to internal contents decondensation, opposite to normal membrane fusion when a full [Ca 2+ ] i signal was generated by caffeine stimulation (with Ca 2+ i and Ca o 2+ available). We conclude the following. (i) Caffeine can mobilize Ca 2+ from cortical stores independent of the presence of Ca o 2+ . (ii) To yield adequate signals for normal exocytosis, Ca 2+ release and Ca 2+ influx both have to occur during caffeine stimulation. (iii) Insufficient [Ca 2+ ] i increase entails caffeinemediated access of Ca 2+ to the secretory contents, thus causing their decondensation before membrane fusion can occur. (iv) Trichocyst decondensation in turn gives a signal for an unusual dissociation of docking/fusion components at the cell membrane. These observations imply different threshold [Ca 2+ ] i -values for membrane fusion and contents discharge.