Conductance studies on trichotoxin_A50E and implications for channel structure.

Trichotoxin_A50E is an 18-residue peptaibol whose crystal structure has recently been determined. In this study, the conductance properties of trichotoxin_A50E have been investigated in neutral planar lipid bilayers. The macroscopic current-voltage curves disclose a moderate voltage-sensitivity and the concentration-dependence suggests the channels are primarily hexameric. Under ion gradients, shifts of the reversal potential indicate that cations are preferentially transported. Trichotoxin displays only one single-channel conductance state in a given experiment, but an ensemble of experiments reveals a distribution of conductance levels. This contrasts with the related peptaibol alamethicin, which produces multiple channel levels in a single experiment, indicative of recruitment of additional monomers into different multimeric-sized channels. Based on these conductance measurements and on the recently available crystal structure of trichotoxin_A50E, which is a shorter and straighter helix than alamethicin, a tightly-packed hexameric model structure has been constructed for the trichotoxin channel. It has molecular dimensions and surface electrostatic potential compatible with the observed conductance properties of the most probable and longer-lived channel.

Ion permeation of pores in model membranes: selectivity, fluctuations and the role of surface charge.

Fluctuation of surface charge on pore walls provides a realistic, additional mechanism for generating fluctuation of ionic current and ionic selectivity in narrow pores.

Understanding the tumor metabolic phenotype in the genomic era.

Now, at the beginning of a new century, 80 years after Warburg's Nobel prize winning discoveries, we are beginning to make sense of the underlying causes of the well known metabolic phenotype of tumor cells. Building on decades of research to understand the interrelationships between respiration and glycolysis in cancer, the tumor metabolic phenotype can now begin to be understood in a genomic context. With the discovery of hypoxia inducible factor-1 (HIF-1), which is widely overexpressed across a broad range of cancers, modern molecular tools have allowed us to put together the pattern of events that might explain the metabolic differences between tumor and normal cells. HIF-1 controls cellular and systemic responses to oxygen availability and coordinates up-regulation of genes involved in many pathways concerned with tumour growth and metabolism including angiogenesis, glucose and energy metabolism, cellular proliferation, differentiation and viability, apoptosis, pH regulation and matrix metabolism. These findings begin to explain how glucose uptake and glycolysis could be up-regulated in cancer cells (through binding to a core DNA recognition sequence) in a co-ordinated and constitutive fashion that may also allow us to elucidate new targets for tumor therapy.

Fluctuation of surface charge in membrane pores.

Surface charge in track-etched polyethylene terephthalate (PET) membranes with narrow pores has been probed with a fluorescent cationic dye (3,3'-diethyloxacarbocyanine iodide (diO-C2-(3))) using confocal microscopy. Staining of negatively charged PET membranes with diO-C2-(3) is a useful measure of surface charge for the following reasons: 1) the dye inhibits K(+) currents through the pores and reduces their selectivity for cations; 2) it inhibits [3H]-choline+ transport and promotes 36Cl- transport across the membrane in a pH- and ionic-strength-dependent fashion; and 3) staining of pores by diO-C2-(3) is reduced by low pH and by the presence of divalent cations such as Ca2+ and Zn2+. Measurement of the time dependence of cyanine staining of pores shows fluctuations of fluorescence intensity that occur on the same time scale as do fluctuations of ionic current in such pores. These data support our earlier proposal that fluctuations in ionic current across pores in synthetic and biological membranes reflect fluctuations in the surface charge of the pore walls in addition to molecular changes in pore proteins.

Evidence-based practice: tap water cleansing of leg ulcers in the community.

To establish and encourage wound management practices based on evidence, a Community Nursing Organization in metropolitan Adelaide began a series of research initiatives in 1997. Based on the results of a wound management survey, and through the processes of participatory action research with clinicians, many wound management practice changes were instigated throughout the Community Nursing Organization. One question remains unanswered: What is the evidence for the use of sterile saline or clean tap water for cleansing of leg ulcers in the community? In this paper we describe a project where we applied the three principles of planning, action and evaluation. Application of these principles enabled clinicians to collaborate in the search for evidence to support or refute tap water cleansing of leg ulcers. To conclude, we report on a pilot research project undertaken to obtain further evidence either to support or refute the use of tap water cleansing for leg ulcers in the community.

A study of membrane activity in rat prostate cancer cells: an evaluation of the FM1-43 dye technique.

A study was initiated to test whether the FM1-43 dye technique could be applied to the study of endocytic membrane activity in two rodent prostate cancer (MAT-LyLu and AT-2) cell lines of markedly different metastatic ability. The lipophilic dye FM1-43, which has frequently been used to monitor endo/exocytic activity in excitable cells was employed. We found, as in excitable tissues, that both strongly metastatic (MAT-LyLu) and weakly metastatic (AT-2) cells in culture take up FM1-43 to give vesicular staining of a variable pattern, which appeared to differ between the two cell lines. However, unlike excitable tissues, neither cell line subsequently released the dye. Indeed, both cell lines retained the dye through several rounds of cell division suggesting that dye incorporated by cells does not enter the endo/exocytotic cycle. Uptake of dye was independent of temperature, Na+/K+ gradients, pH or metabolism. We suggest that passive accumulation of FM1-43 can occur in cancer cells and should not, automatically, be interpreted as evidence of endocytosis.

Causes and consequences of tumour acidity and implications for treatment.

Tumour cells have a lower extracellular pH (pHe) than normal cells; this is an intrinsic feature of the tumour phenotype, caused by alterations either in acid export from the tumour cells or in clearance of extracellular acid. Low pHe benefits tumour cells because it promotes invasiveness, whereas a high intracellular pH (pHi) gives them a competitive advantage over normal cells for growth. Molecular genetic approaches have revealed hypoxia-induced coordinated upregulation of glycolysis, a potentially important mechanism for establishing the metabolic phenotype of tumours. Understanding tumour acidity opens up new opportunities for therapy.

Thermal control of drug release by a responsive ion track membrane observed by radio tracer flow dialysis.

The combination of a responsive hydrogel with a rigid porous supporting structure yield a membrane with high mechanical strength and high on-off-permeability ratio. A membrane consisting of an ion track filter with a thermally responsive lining was prepared by penetrating a 19 micron thick foil of poly(ethylene terephthalate) (PET) with swift heavy ions at a fluence of 5 x 10(5) ions/cm2, followed by etching of the ion tracks to generate an ion track filter with 2.9 micron pore diameter, onto which a thin layer of poly(N-isopropylacrylamide) (NIPAAm) hydrogel was grafted. It was revealed that the mass flow of various molecules (water, chloride-, choline+, insulin, and albumin) through the membrane could be thermally controlled. The on-off-permeability ratio ranged between 3 and 10 increasing with molecular weight. Over a storage time of 5 months the permeabilities varied up to a factor of 2.6, while the on-off-permeability ratio and temperature sensitivity remained practically constant.

Influence of pH on the uptake of 5-fluorouracil into isolated tumour cells.

To investigate the possible dependence of 5-fluorouracil (5FU) uptake in tumours on the intra- (pHi) and extracellular (pHe) pH, a pH gradient (deltapH) was imposed across the plasma membrane of ascites tumour cells in vitro, similar to that known to occur in some solid tumours in vivo, by incubation in media of PHe 5-8. A > or = 2:1 (intracellular/extracellular) accumulation of radiolabelled 5FU occurred after 5 min incubation of the cells with 0.5 mM 5FU at pHe of 5.0, 5.5 or 6.0. 5FU metabolism is slow under these conditions, and 5FU uptake was not affected by longer incubations up to 20 min, nor by the absence of a sodium gradient. pHi was estimated from the distribution of the weak acid, 5.5-dimethyl-2,4-oxazolidione ([14C]DMO) across the cell membrane. There was significant correlation between the intracellular/extracellular 5FU ratio and pHe (from pHe 6-8), deltapH and pHi (P < 0.02). Similar results were obtained with HT29 cells. Incubation with a drug that made plasma membranes permeable to H+ significantly decreased 5FU uptake in Lettre cells. The co-transport of 5FU may occur on a proton symport using the proton motive force of the deltapH.

A conserved tryptophan in pneumolysin is a determinant of the characteristics of channels formed by pneumolysin in cells and planar lipid bilayers.

Pneumolysin is one of the family of thiol-activatable, cytolytic toxins. Within these toxins the amino acid sequence Trp-Glu-Trp-Trp is conserved. Mutations made in this region of pneumolysin, residues 433-436 inclusive, did not affect cell binding or the formation of toxin oligomers in the target cell membrane. However, the mutations did affect haemolysis, leakage of low-molecular-mass metabolites from Lettre cells and the induction of conductance channels across planar lipid bilayers. Of eight modified pneumolysins examined, Trp-433-->Phe showed the smallest amount of haemolysis or leakage (less than 5% of wild type). Pneumolysin-induced leakage from Lettre cells was sensitive to inhibition by bivalent cations but the extent of inhibition varied depending on the modification. Leakage by the mutant Trp-433-->Phe was least sensitive to cation inhibition. The ion-conducting channels formed across planar lipid bilayers exhibit small (less than 30 pS), medium (30 pS-1 nS) and large (more than 1 nS) conductance steps. Small- and medium-sized channels were preferentially closed by bivalent cations. In contrast with wild-type toxin, which formed predominantly small channels, the modified toxin Trp-433-->Phe formed large channels that were insensitive to cation-induced closure. Polysaccharides of molecular mass more than 15 kDa inhibited haemolysis by wild-type toxin, but polysaccharide of up to 40 kDa did not prevent haemolysis by Trp-433-->Phe. Electron microscopy revealed that Trp-433-->Phe formed oligomeric arc and ring structures with dimensions identical with those of wild-type toxin, and that the ratio of arcs to rings formed was the same for wild-type toxin and the Trp-433-->Phe variant. We conclude that the change Trp-433-->Phe affects channel formation at a point subsequent to binding to the cell membrane and the formation of oligomers, and that the size of arc and ring structures revealed by electron microscopy does not reflect the functional state of the channels.

Structure-based prediction of the conductance properties of ion channels.

The HOLE procedure allows the prediction of the absolute conductance of an ion channel model from its structure. The original prediction method uses an empirically corrected Ohmic method. It is most successful, with predictions being reliable to within a factor of two. A new modification of the procedure is presented in which the self-diffusion coefficients of water molecules from molecular dynamics simulation are used to replace the empirical correction factor. A "prediction" of the conductance for the porin OmpF by the new method is made and shown to be very close to the experimental value. HOLE also allows the prediction of the effect that the addition of non-electrolyte polymers will have on channel conductance. The method has great potential to yield structural information from data provided by single channel recordings but needs further validation by making measurements on channels of known structure. Preliminary results are given of single channel records establishing the effects of non-electrolytes on the conductance of gramicidin D channels. As an example of the potential uses of the procedure application is made to examine the oligomerization of alpha-toxin (alpha-hemolysin) channels. A model for the alpha-toxin hexamer, based on the crystal structure for the heptamer, is generated using molecular mechanics methods. The compatibility of the structures with single channel conductance data is assessed using HOLE.

Specialized scanning ion-conductance microscope for imaging of living cells.

A specialized scanning ion conductance microscope (SICM) for imaging living cells has been developed from a conventional patch-clamp apparatus, which uses a glass micropipette as the sensitive probe. In contrast with other types of scanning probe microscope, the SICM probe has significant advantages for imaging living cells: it is most suitable for imaging samples immersed in water solutions; and since the probe senses ion current and does not need physical contact with the sample during the scan, any preliminary preparation of cells (fixation or adherence to a substrate) is unnecessary. We have successfully imaged murine melanocytes in growth medium. The microscope images the highly convoluted surface structures without damaging or deforming them, and reveals the true, three-dimensional relief of the cells. This instrument has considerable ability to operate, potentially simultaneously, in applications as diverse as real-time microscopy, electrophysiology, micromanipulation and drug delivery.

Scanning ion conductance microscopy of living cells.

Currently there is a great interest in using scanning probe microscopy to study living cells. However, in most cases the contact the probe makes with the soft surface of the cell deforms or damages it. Here we report a scanning ion conductance microscope specially developed for imaging living cells. A key feature of the instrument is its scanning algorithm, which maintains the working distance between the probe and the sample such that they do not make direct physical contact with each other. Numerical simulation of the probe/sample interaction, which closely matches the experimental observations, provides the optimum working distance. The microscope scans highly convoluted surface structures without damaging them and reveals the true topography of cell surfaces. The images resemble those produced by scanning electron microscopy, with the significant difference that the cells remain viable and active. The instrument can monitor small-scale dynamics of cell surfaces as well as whole-cell movement.

A novel explanation for fluctuations of ion current through narrow pores.

Fluctuation of ion current, between a high conductance and a low conductance state, through biological ion channels and pores is assumed to arise from conformational changes between an "open" and a "closed" configuration. Here we offer an additional mechanism that arises from changes in ionization of fixed charges within, or at the mouth of, a channel or pore. Our hypothesis, which is based on measurements of ion selectivity alongside ion current, applies to pores through some synthetic membranes and through channels-such as those created by certain toxins-that remain (at least partially) open in the low conductance state. It may also explain the phenomena of "open channel noise" and "substate behavior" that characterize several endogenous ion channels and should be considered when modeling the behavior of such channels.

Feigned mental disorder in prisoners referred to forensic mental health services.

This paper describes a study that aimed to identify the extent to which feigning of mental illness represents a significant problem in prisoners referred to a medium secure unit. A clinical method was used to assess the prisoners, employing the Structured Interview of Reported Symptoms (SIRS) and selected Minnesota Multiphasic Personality Inventory-2 (MMPI-2) validity indices. Stringent criteria were used to classify 60 consecutive referrals to a secure unit as feigning mental disorder or not. In addition, the study examined characteristic differences, on a number of selected variables, between those prisoners who feign and those who respond honestly. The personality profiles of prisoners who feign were also investigated. Analysis of data showed that 32% of the sample (P < 0.01) could be classified as fabricating or exaggerating symptoms of mental illness. It is suggested that such presentations may be more prevalent than previously considered. Few differences were observed between the groups on selected variables, although prisoners who feigned mental illness demonstrated significantly higher dependent and anxious (avoidant) personality types. The implications of the findings are discussed in relation to available models of feigned presentations.

31P-magnetic resonance spectroscopy studies of nucleated and non-nucleated erythrocytes; time domain data analysis (VARPRO) incorporating prior knowledge can give information on the binding of ADP.

Human erythrocytes have no nucleus, mitochondria or endoplasmic reticulum, whereas chicken erythrocytes have a nucleus and mitochondria and are closer in internal morphology, to cells such as the hepatocyte. Erythrocytes were used to test the hypothesis that 31P-MRS invisibility of ADP is associated with the presence of intracellular organelles. Simple frequency domain spectral analysis methods showed that all the acid extractable ADP (and ATP) was MR-visible in human erythrocytes. However, such methods gave variable estimates for 31P-NMR spectra of fresh chicken erythrocytes from which no conclusions could be drawn about the MR-visibility of ADP. Only when the data were fitted by a method incorporating prior knowledge of the ATP and ADP peak structure, using the time domain VARPRO method, was it possible to conclude that in fresh chicken erythrocytes, similar to other nucleated cells (liver, muscle), all the acid extractable ADP appeared to be MRS invisible, indicating binding or sequestration by intracellular organelles.

Diffusion through narrow pores: movement of ions, water and nonelectrolytes through track-etched PETP membranes.

The rates at which ions (86Rb+, [3H]-choline, 36Cl), 3H2O and nonelectrolytes ([14C]-urea, [14C]-glycerol, and [14C]-sugars) equilibrate across track-etched polyethyleneterephthalate (PETP) membranes (isotopic diffusion) have been measured by a 'static' and a 'dynamic' technique under conditions where no net flow takes place; the two techniques give essentially the same results. All tracers diffuse faster the longer the membranes are etched, consistent with an increase in pore size. Water and neutral solutes diffuse at rates that are relatively independent of ionic strength, pH or the presence of divalent cations. Diffusion of cations is decreased by high ionic strength, by reducing pH or by addition of divalent cations; diffusion of chloride is increased by these procedures. Treatment of the membrane with diazomethane to reduce the negative fixed charge decreases diffusion of cations and increases that of anions; diffusion of water and neutral solutes is unaffected by methylation except in the membranes with the narrowest pores (i.e., those etched for the shortest time), in which case diffusion is reduced. We conclude (1) that the special features of flow near a charged surface apply to ions but not to water or nonelectrolytes and (2) that calculation of absolute rates of diffusion leads to values for the radii of pores through track-etched PETP membranes that are in remarkably good agreement with measured values.