Public views of the benefits and barriers to the consumption of a plant-based diet.

OBJECTIVE: The aim of this study was to examine consumers' perceived benefits and barriers to the consumption of a plant-based diet. DESIGN: Mail survey that included questions on perceived benefits and barriers to the consumption of a plant-based diet. SETTING: Victoria, Australia. SUBJECTS: Four hundred and fifteen randomly selected Victorian adults. RESULTS: The main perceived barrier to adoption of a plant-based diet was a lack of information about plant-based diets (42% agreement). Sex, age and education differences were present in over a quarter of the barrier items. For example, non-university-educated respondents and older people were less willing to change their current eating pattern than were university educated and younger respondents. The main benefits associated with plant-based diets were health benefits, particularly decreased saturated fat intake (79% agreement), increased fibre intake (76%), and disease prevention (70%). Age, sex and education differences with regard to benefits were apparent, although sex differences were more important than age or education differences. CONCLUSIONS: The majority of respondents perceived there to be health benefits associated with the consumption of a plant-based diet. Compared with the proportion of respondents who agreed that there were particular benefits of eating a plant-based diet, perceived barriers were relatively low. An understanding of the perceived benefits and barriers of consuming a plant-based diet will help formulate strategies that aim to influence beliefs about plant foods, plant food consumption, and, ultimately, public health.

Consumers' readiness to eat a plant-based diet.

OBJECTIVE: The aim of this study was to examine consumers' readiness to change to a plant-based diet. DESIGN: Mail survey that included questions on readiness to change, eating habits and perceived benefits and barriers to the consumption of a plant-based diet. SETTING: Victoria, Australia. SUBJECTS: A total of 415 randomly selected adults. RESULTS: In terms of their readiness to eat a plant-based diet, the majority (58%) of participants were in the precontemplation stage of change, while 14% were in contemplation/preparation, and 28% in action/maintenance. Those in the action/maintenance stage ate more fruit, vegetables, nuts, seeds, whole-meal bread, and cooked cereals than those in earlier stages. There were statistically significant differences in age and vegetarian status between the stages of change, but not for other demographic variables. There were strong differences across the stages of change with regard to perceived benefits and barriers to plant-based diets. For example, those in action/maintenance scored highest for benefit factors associated with well-being, weight, health, convenience and finances, whereas those in the precontemplation stage did not recognise such benefits. CONCLUSIONS: These findings can be utilised to help provide appropriate nutrition education and advertising, targeted at specific stages of change. For example, education about how it is possible to obtain iron and protein from a plant-based diet and on the benefits of change, in addition to tips on how to make a gradual, easy transition to a plant-based diet, could help progress precontemplators to later stages. SPONSORSHIP: Australian Research Council.

Interaction of luminal calcium and cytosolic ATP in the control of type 1 inositol (1,4,5)-trisphosphate receptor channels.

Ca(2+) within intracellular stores (luminal Ca(2+)) is believed to play a role in regulating Ca(2+) release into the cytosol via the inositol (1,4,5)-trisphosphate (Ins(1,4,5)P(3))-gated Ca(2+) channel (or Ins(1,4,5)P(3) receptor). To investigate this, we incorporated purified Type 1 Ins(1,4,5)P(3) receptor from rat cerebellum into planar lipid bilayers and monitored effects at altered luminal [Ca(2+)] using K(+) as the current carrier. At a high luminal [Ca(2+)] and in the presence of optimal [Ins(1,4,5)P(3)] and cytosolic [Ca(2+)], a short burst of Ins(1,4,5)P(3) receptor channel activity was followed by complete inactivation. Lowering the luminal [Ca(2+)] caused the channel to reactivate indefinitely. At luminal [Ca(2+)], reflecting a partially empty store, channel activity did not inactivate. The addition of cytosolic ATP to a channel inactivated by high luminal [Ca(2+)] caused reactivation. We provide evidence that luminal Ca(2+) is exerting its effects via a direct interaction with the luminal face of the receptor. Activation of the receptor by ATP may act as a device by which cytosolic Ca(2+) overload is prevented when the energy state of the cell is compromised.

Expression of a gene for a porin-like protein of the OmpA family from Mycobacterium tuberculosis H37Rv.

An open reading frame in the genomic database of Mycobacterium tuberculosis H37Rv was identified as having homology with an outer membrane protein. We found that the gene specified a protein belonging to the OmpA family, which includes some porins of gram-negative organisms. The gene was amplified by PCR and cloned into Escherichia coli. Overexpression of the gene was toxic to the host, but limited amounts could be purified from cells before growth ceased. A truncated gene devoid of the code for a presumed signal sequence was well expressed, but the protein had no pore-forming activity in the liposome swelling assay. However, the intact protein, OmpATb, behaved as a porin of low specific activity, with a pore diameter of 1.4 to 1.8 nm, and was also active in planar lipid bilayers, showing a single-channel conductance of 700 pS. The protein had a molecular mass of about 38 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A polyclonal rabbit antiserum raised to the truncated protein recognized a protein of similar molecular mass in detergent extracts of broken M. tuberculosis cells. Reverse transcription-PCR confirmed that the gene for OmpATb was expressed in M. tuberculosis cells growing in culture. Comparison of the purified protein with that in the detergent-extracted preparation using liposomes and planar lipid bilayers showed that the two materials had similar pore-forming properties. OmpATb is different from either of the mycobacterial porins described so far. This is the first report of a porin-like molecule from M. tuberculosis; the porin is likely to be important in controlling the access of hydrophilic molecules to the bacterial cell.

The effects of free [Ca2+] on the cytosolic face of the inositol (1,4,5)-trisphosphate receptor at the single channel level.

Cytosolic free Ca2+ has been shown to have both activating and inhibitory effects upon the inositol (1,4,5) trisphosphate receptor (InsP3R) during intracellular Ca2+ release. The effects of cytosolic free Ca2+ on the InsP3R have already been monitored using cerebellar microsomes (containing InsP3R) incorporated into planar lipid bilayers [Bezprozvanny, Watras and Ehrlich (1991) Nature (London) 351, 751-754]. In these experiments the open probability of the channel exhibited a 'bell-shaped Ca2+ dependence'. However, this has only been seen when the receptor is in the presence of its native membrane (e.g. microsomal vesicles). Using solubilized, purified InsP3R incorporated into planar lipid bilayers using the 'tip-dip' technique, investigations were carried out to see if the same effect was seen in the absence of the native membrane. Channel activity was observed in the presence of 4 microM InsP3 and 200 nM free Ca2+. Mean single channel current was 2.69 pA and more than one population of lifetimes was observed. Two populations had mean open times of approx. 9 and 97 ms. Upon increasing the free [Ca2+] to 2 microM, the mean single channel current decreased slightly to 2.39 pA, and the lifetimes increased to 30 and 230 ms. Elevation of free -Ca2+- to 4 microM resulted in a further decrease in mean single channel current to 1.97 pA as well as a decrease in lifetime to approx. 8 and 194 ms. At 10 microM free [Ca2+] no channel activity was observed. Thus, with purified receptor in artificial bilayers, free [Ca2+] on the cytosolic face of the receptor has major effects on channel behaviour, particularly on channel closure, although inhibition of channel activity is not seen until very high free [Ca2+] is reached.

Voltage-gating of Escherichia coli porin: a cystine-scanning mutagenesis study of loop 3.

Porins, such as Escherichia coli OmpF, provide the only reported example of a voltage-gated channel where the three-dimensional structure is known to high resolution. Mutations that affect voltage-gating are clustered around the eyelet region, which is a mid-channel constriction caused by a polypeptide loop (L3) folding inside the lumen of this beta-barrel pore. These data, combined with molecular dynamics simulations, indicate that voltage-gating may involve L3 displacement. We have constructed six double cysteine OmpF mutants, five of which form disulphide bonds fixing L3 in the conformation determined by X-ray crystallography. These channels have altered single-channel conductances but unimpaired voltage-gating. The data show that L3 movement is not required for voltage-gating.

Brucella Omp2a and Omp2b porins: single channel measurements and topology prediction.

Brucella usually carry two highly homologous genes (omp2a and omp2b) for porin-like proteins. In several B. abortus biovars the omp2a gene has a large deletion compared to other Brucella omp2's. In this study we have measured Omp2 pore activity in planar bilayers. Omp2b exhibits well-defined trimeric channel activity whilst Omp2a forms monomeric pores of variable size which are smaller than Omp2b. No sequence homology exists between Omp2 and porins of known structure, so hydrophobic moment analysis has been used to model their membrane topology. From this it appears likely that the deletion removes the crucial L3 internal loop.

Lateral diffusion in planar lipid bilayers: a fluorescence recovery after photobleaching investigation of its modulation by lipid composition, cholesterol, or alamethicin content and divalent cations.

In spite of the fact that planar lipid bilayers are still the best-suited artificial membrane system for the study of reconstituted ion channels and receptors, data dealing with their physical characterization, especially as regards dynamics, are scanty. A combined electrical and optical chamber was designed and allowed fluorescence recovery after photobleaching recovery curves to be recorded from stable virtually solvent-free bilayers. D, the lateral diffusion coefficient of N-(7-nitrobenzoyl-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn- glycero-3-phosphoethanolamine, was found to be relatively insensitive to the phospholipid composition (headgroup, chain unsaturation, etc.), whereas inclusion of 33-50% cholesterol in the membrane reduced D by a factor of 2. Divalent cations significantly reduced D of negatively charged bilayers. These results compare well with data gathered on other model and natural systems. In addition, the incorporation of the voltage-dependent pore-former alamethicin did slightly reduce lipid lateral mobility. This study demonstrates the feasibility of such experiments with planar bilayers, which are amenable to physical constraints, and thus offers new opportunities for systematic studies of structure-function relationships in membrane-associating molecules.

The inositol 1,4,5-trisphosphate-gated Ca2+ channel: effect of the protein thiol reagent thimerosal on channel activity.

The solubilized partially purified Ins(1,4,5)P3-sensitive Ca2+ channel from rat cerebellum has been reconstituted into planar lipid bilayer membranes by the 'tip-dip' method [Ehrlich (1992) Methods Enzymol. 207, 463-471] allowing low noise current records. Single-channel events have been recorded. In the presence of 10 microM Ins(1,4,5)P3, 50 microM ATP, and 0.2 microM Ca2+ the Ins(1,4,5)P3 receptor channel opens to a conductance level of 53 pS. In the presence of 100 microM thimerosal (TMS), a sulphydryl-oxidizing agent, three subconductance levels (60 pS, 80 pS and 120 pS) were observed. More than one population of mean open times was found, both in the absence and presence of TMS, although TMS affected the length of the open time by decreasing the short opening significantly from 4.05 ms to 2.78 ms and increasing the longer open time from 27.8 ms to 94.8 ms. The results indicate that TMS enhances Ins(1,4,5)P3-induced Ca2+ release by both altering the open times of the channel significantly and causing a shift to higher subconductance levels.

Altered voltage sensitivity of mutant OmpC porin channels.

Single OmpC porin channels have been reconstituted in planar bilayer membranes. Wild-type OmpC forms trimers which are largely insensitive to voltages below 250 mV.A single-point mutation of the ompC gene has been prepared resulting in replacement of Trp56 by Cys in the eyelet region of the channel wall in a highly conserved segment of the polypeptide. The monomeric channels of which the trimer is composed have smaller conductivity in 1 M NaCl (400 +/- 20 pS, mean and S.E.M., n=30) and increased voltage sensitivity by comparison with the wild-type under similar conditions, whereas other (Dex) mutants form larger channels and display different behaviour. Further, by treatment in SDS solutions at different temperatures, the W56C mutant has been shown to be less stable than either the wild-type or the Dex mutants.

Main intrinsic polypeptide and water movement--recent developments and future prospects.

Age-related lens opacity is the major cause of loss of vision affecting more than half of the world's blind. The development of cataract is associated with changes in the structure of the lens. The lens consists largely of closely packed fibre cells forming a transparent syncytium. The main intrinsic polypeptide (MIP) of the lens fibre cells forms 40% of the fibre membrane, and forms a system of membrane channels. The precise structure and function of these channels remains obscure, although their role as large water-filled channels has been suggested in maintenance of structural integrity as well as their role in transport of nutrients. MIP is a member of a large family of channels of common ancestry. Some of the most recent studies of structure and function of this family have been carried out on CHIP (a water channel found in red blood cells) which is a member of this family closely related to MIP. Both CHIP and MIP m-RNA's have been expressed in oocyte membranes, and water permeabilities per channel have been measured. Surprisingly, CHIP and MIP channels have high and low water permeabilities, respectively. This is discussed in terms of recent high resolution structure determinations, the lens fibre MIP function, and future studies.

The nodulation-signaling protein NodO from Rhizobium leguminosarum biovar viciae forms ion channels in membranes.

The secreted nodulation-signaling protein NodO was purified from the supernatant of cultures of Rhizobium leguminosarum biovar viciae. The native protein has a M(r) of approximately 67,000, suggesting that it exists as a dimer since the DNA sequence predicts a M(r) of 30,002. Pure NodO protein had no protease, pectinase, or cellulase activity, and no binding was observed to lipooligosaccharide nodulation factors. Although NodO is relatively hydrophilic, it appeared to insert into liposomes and was protected by liposomes from proteolytic cleavage. When added to planar lipid bilayers, NodO formed cation-selective channels that allowed the movement of monovalent cations (K+ and Na+) across the membrane. NodO is a Ca(2+)-binding protein; in the presence of high concentrations of Ca2+, channel activity was reduced. We hypothesize that NodO plays a role in nodulation signaling by stimulating uptake of nodulation factors or by forming cation-specific channels that function synergistically with the proposed lipooligosaccharide-induced depolarization of the plasma membrane of leguminous plants.

Characterisation of the porin of Rhodobacter capsulatus 37b4 in planar lipid bilayers.

The outer membrane of Gram-negative bacteria contains aqueous channels, porins, which aid the diffusion of small hydrophilic molecules across it. Escherichia coli, as enteric bacteria, are able to survive a hostile environment of proteases, surfactants, and drastic changes of osmotic pressure. Rhodobacter capsulatus is not an enteric bacterium and as such has not evolved to resist the same challenges. Porins, which have molecular weight of approximately 35 kDa, form trimeric channels with a solute exclusion limit of about 600 Da. Most of them open and close in a controlled manner as a function of p.d. This function is little understood at present. The functional properties of single trimers of the major porin of Rhodobacter capsulatus 37b4 have been investigated in planar artificial bilayers. On application of a suitable p.d. the observed trimer closes in approximately three equal steps. The behaviour is completely symmetrical as regards closure in response to p.d.'s of opposite polarity and is strongly cation selective.

ompC mutants which allow growth on maltodextrins show increased channel size and greater voltage sensitivity.

Misra and Benson [(1988) J. Bacteriol. 170, 3611-3617] showed that point mutations in the ompC gene can allow Escherichia coli to grow on maltotriose in the absence of LamB. This report shows that these mutants produce OmpC porins with increased single channel conductance compared to the wild type. The mutants showed similar voltage dependence to each other and to PhoE by being totally closed at 200 mV. The wild type from various sources was largely insensitive to voltages below 200 mV and thus 6 point mutations at 3 sites appear to increase the voltage dependence of OmpC channels.

Voltage-dependent block of anthrax toxin channels in planar phospholipid bilayer membranes by symmetric tetraalkylammonium ions. Single-channel analysis.

Previous studies have shown that symmetric tetraalkylammonium ions affect, in a voltage-dependent manner, the conductance of membranes containing many channels formed by the PA65 fragment of anthrax toxin. In this paper we analyze this phenomenon at the single-channel level for tetrabutylammonium ion (Bu4N+). We find that Bu4N+ induces a flickery block of the PA65 channel when present on either side of the membrane, and this block is relieved by large positive voltages on the blocking-ion side. At high frequencies (greater than 2 kHz) we have resolved individual blocking events and measured the dwell times in the blocked and unblocked states. These dwell times have single-exponential distributions, with time constants tau b and tau u that are voltage dependent, consistent with the two-barrier, single-well potential energy diagram that we postulated in our previous paper. The fraction of time the channel spends unblocked [tau u/(tau u + tau b)] as a function of voltage is identical to the normalized conductance-voltage relation determined from macroscopic measurements of blocking, thus demonstrating that these single channels mirror the behavior seen with many (greater than 10,000) channels in the membrane. In going from large negative to large positive voltages (-100 to +160 mV) on the cis (PA65-containing) side of the membrane, one sees the mean blocked time (tau b) increase to a maximum at +60 mV and then steadily decline for voltages greater than +60 mV, thereby clearly demonstrating that Bu4N+ is driven through the channel by positive voltages on the blocking-ion side. In other words, the channel is permeable to Bu4N+. An interesting finding that emerges from analysis of the voltage dependence of mean blocked and unblocked times is that the blocking rate, with Bu4N+ present on the cis side of the membrane, plateaus at large positive cis voltages to a voltage-independent value consistent with the rate of Bu4N+ entry into the blocking site being diffusion limited.

Changes in platelet membrane fatty acids after myocardial infarction.

Myocardial infarction (MI) is a common cause of morbidity and mortality, and platelets may contribute to its development. Platelet membrane composition can influence platelet function. In this study changes in platelet membrane fatty acids with time following MI were explored. Platelet membrane fatty acid profiles were studied in 40 patients after MI, at a mean of 8 days, and compared with a control group of 17 subjects awaiting minor surgery. They were restudied at 1 month and 3 months. Significant changes were found within the patient group in 18:1, which fell with time (19.99% +/- 1.24 to 18.73% +/- 1.16 at 3 months, P less than 0.001), 22:3 + 24:1, which also fell (1.97% +/- 0.48 to 1.46% +/- 0.49, P less than 0.001), and in 18:2, which increased (3.92% +/- 0.77 to 5.04% +/- 1.15 P less than 0.001). Comparison with controls showed no significant differences at baseline, a small increase in 22:4 at 1 month and 3 months in MI group and a decrease in 22:3 + 24:1 in MI group at 3 months. No changes were noted in 20:4, 20:5 or in the polyunsaturated/saturated fatty acid ratio. The explanation for these findings is not known. The possible influence of diet and other factors is discussed.

Lipids and platelet function in runners.

The effect of distance running on the fatty acid composition of platelet and erythrocyte membranes has been investigated, together with platelet aggregation and levels of high density lipoprotein-cholesterol (HDLc), low density lipoprotein cholesterol (LDLc), total cholesterol and triglycerides in runners (n = 11) and healthy age-matched non-running controls (n = 12). Platelet aggregation and fatty acid composition of membrane lipids in both platelets and erythrocytes are similar in both groups with the following exceptions: in platelets docosahexaenoic acid (C22:6 omega 3) is significantly higher in runners; in erythrocytes the fatty acids C20:3 omega 6/C22:1 omega 9 and C22:5 omega 3 are significantly higher in runners. There were no significant differences between the levels of HDLc, LDLc and total cholesterol in runners and controls although triglycerides were significantly lower in runners. Possible beneficial effects of running are probably mediated through effects on serum lipid and lipoprotein concentrations, and probably not due to any antithrombotic effect of platelets.

The role of acyl chain character and other determinants on the bilayer activity of A21978C an acidic lipopeptide antibiotic.

An acidic lipopeptide A21978C has previously been shown to have a powerful antibiotic activity against Gram-positive organisms. Due to its ability to increase the K+ permeability of bacterial cells and its specific calcium requirement, which is similar to a previously described ionophore CDA, its effect on planar bilayer membranes has been studied. Although it produces significant increases in the conductivity of lipid bilayers it is shown that this alone cannot account for its in vivo activity. Similarly, unlike the in vivo results, the Ca2+-induced increases in bilayer conductivity can be mimicked by Mg2+ and charged lipids. Results from a series of homologues differing in the length of the acyl moiety show a close similarity between bilayer conductance and LD50 trends from in vivo studies. A complex activity is proposed which depends upon incorporation in, rather than disruption of, the bilayer membrane.

Characterisation of channels induced in planar bilayer membranes by detergent solubilised Escherichia coli porins.

Purified OmpF, OmpC, NmpC, PhoE and Lc (Protein 2) porins from the Escherichia coli outer membrane were incorporated into planar phospholipid bilayer membranes and the permeability properties of the pores studied. Triton X-100 solubilised porin samples showed large and reproducible increases in membrane conductivity composed of discreet single-channel events. The magnitude of the cation selectivity found for the porins was in the order OmpC greater than OmpF greater than NmpC = Lc; PhoE was anion selective. For the cation selective porins the cation/anion permeability ratios in a variety of solutes ranged from 6 to 35. Further information on the internal structure of the porins was obtained by examination of the single-channel conductance and this was used to interpret macroscopic observations and to estimate single-channel diameters. The same porins solubilised in SDS exhibited slight conductance increase with no observable single-channel activity. Use of on-line microcomputer techniques confirmed the ohmic current vs. voltage behaviour for all the single porin channels examined.

A new channel-forming antibiotic from Streptomyces coelicolor A3(2) which requires calcium for its activity.

A recently discovered antibiotic (CDA; calcium-dependent antibiotic) of Streptomyces coelicolor A3(2) was found to be effective against a wide range of Gram-positive bacteria only in the presence of calcium ions. Producer and non-producer strains were identified and several media tested for their ability to support antibiotic production. The action of calcium was not simulated by any of the other cations tested. The antibiotic was found to induce discrete conductance fluctuations in planar lipid bilayer consistent with a channel-forming action. The electrical potential difference caused by a concentration difference of various salts across the CDA-containing bilayer, showed the channel to be cation-selective but of a size that discriminated against tetramethyl ammonium and choline ions. The data indicate that the antibiotic activity of CDA is due to its action as a calcium-dependent ionophore.