A comparison of the self-association behavior of the plant cyclotides kalata B1 and kalata B2 via analytical ultracentrifugation

The recently discovered cyclotides kalata B1 and kalata B2 are miniproteins containing a head-to-tail cyclized backbone and a cystine knot motif, in which disulfide bonds and the connecting backbone segments form a ring that is penetrated by the third disulfide bond. This arrangement renders the cyclotides extremely stable against thermal and enzymatic decay, making them a possible template onto which functionalities can be grafted.We have compared the hydrodynamic properties of two prototypic cyclotides, kalata B1 and kalata B2, using analytical ultracentrifugation techniques. Direct evidence for oligomerization of kalata B2 was shown by sedimentation velocity experiments in which a method for determining size distribution of polydisperse molecules in solution was employed. The shape of the oligomers appears to be spherical. Both sedimentation velocity and equilibrium experiments indicate that in phosphate buffer kalata B1 exists mainly as a monomer, even at millimolar concentrations. In contrast, at 1.6 mM, kalata B2 exists as an equilibrium mixture of monomer (30%), tetramer (42%), octamer (25%), and possibly a small proportion of higher oligomers. The results from the sedimentation equilibrium experiments show that this self-association is concentration dependent and reversible. We link our findings to the three-dimensional structures of both cyclotides, and propose two putative interaction interfaces on opposite sides of the kalata B2 molecule, one involving a hydrophobic interaction with the Phe(6), and the second involving a charge-charge interaction with the Asp(25) residue. An understanding of the factors affecting solution aggregation is of vital importance for future pharmaceutical application of these molecules.

Estimating the potential refolding yield of recombinant proteins expressed as inclusion bodies

Recombinant protein production in bacteria is efficient except that insoluble inclusion bodies form when some gene sequences are expressed. Such proteins must undergo renaturation, which is an inefficient process due to protein aggregation on dilution from concentrated denaturant. In this study, the protein-protein interactions of eight distinct inclusion-body proteins are quantified, in different solution conditions, by measurement of protein second virial coefficients (SVCs). Protein solubility is shown to decrease as the SVC is reduced (i.e., as protein interactions become more attractive). Plots of SVC versus denaturant concentration demonstrate two clear groupings of proteins: a more aggregative group and a group having higher SVC and better solubility. A correlation of the measured SVC with protein molecular weight and hydropathicity, that is able to predict which group each of the eight proteins falls into, is presented. The inclusion of additives known to inhibit aggregation during renaturation improves solubility and increases the SVC of both protein groups. Furthermore, an estimate of maximum refolding yield (or solubility) using high-performance liquid chromatography was obtained for each protein tested, under different environmental conditions, enabling a relationship between yield and SVC to be demonstrated. Combined, the results enable an approximate estimation of the maximum refolding yield that is attainable for each of the eight proteins examined, under a selected chemical environment. Although the correlations must be tested with a far larger set of protein sequences, this work represents a significant move beyond empirical approaches for optimizing renaturation conditions. The approach moves toward the ideal of predicting maximum refolding yield using simple bioinformatic metrics that can be estimated from the gene sequence. Such a capability could potentially screen, in silico, those sequences suitable for expression in bacteria from those that must be expressed in more complex hosts. (C) 2004 Wiley Periodicals, Inc.

Operational regimes for a simplified one-step artificial chaperone refolding method

The artificial chaperone method for protein refolding developed by Rozema et al. (Rozema, D.; Gellman, S. H. J. Am. Chem. Soc. 1995, 117 (8), 2373-2374) involves the sequential dilution of denatured protein into a buffer containing detergent (cetyltrimethylammonium bromide, CTAB) and then into a refolding buffer containing cyclodextrin WD). In this paper a simplified one-step artificial chaperone method is reported, whereby CTAB is added directly to the denatured solution, which is then diluted directly into a refolding buffer containing P-cyclodextrin (P-CD). This new method can be applied at high protein concentrations, resulting in smaller processing volumes and a more concentrated protein solution following refolding. The increase in achievable protein concentration results from the enhanced solubility of CTAB at elevated temperatures in concentrated denaturant. The refolding yields obtained for the new method were significantly higher than for control experiments lacking additives and were comparable to the yields obtained with the classical two-step approach. A study of the effect of beta-CD and CTAB concentrations on refolding yield suggested two operational regimes: slow stripping ( beta-CDXTABsimilar to1), most suited for higher protein concentrations, and fast stripping (beta-CD/CTABsimilar to2.7), best suited for lower protein concentrations. An increased chaotrope concentration resulted in higher refolding yields and an enlarged operational regime.

Interpretation of negative values of the interaction parameter in the adsorption equation through the effects of surface layer heterogeneity

The problem of the negative values of the interaction parameter in the equation of Frumkin has been analyzed with respect to the adsorption of nonionic molecules on energetically homogeneous surface. For this purpose, the adsorption states of a homologue series of ethoxylated nonionic surfactants on air/water interface have been determined using four different models and literature data (surface tension isotherms). The results obtained with the Frumkin adsorption isotherm imply repulsion between the adsorbed species (corresponding to negative values of the interaction parameter), while the classical lattice theory for energetically homogeneous surface (e.g., water/air) admits attraction alone. It appears that this serious contradiction can be overcome by assuming heterogeneity in the adsorption layer, that is, effects of partial condensation (formation of aggregates) on the surface. Such a phenomenon is suggested in the Fainerman-Lucassen-Reynders-Miller (FLM) 'Aggregation model'. Despite the limitations of the latter model (e.g., monodispersity of the aggregates), we have been able to estimate the sign and the order of magnitude of Frumkin's interaction parameter and the range of the aggregation numbers of the surface species. (C) 2004 Elsevier B.V All rights reserved.

Translational incorporation of L-3,4-dihydroxyphenylalanine into proteins

An Escherichia coli cell-free transcription/translation system was used to explore the high-level incorporation Of L-3,4-dihydroxyphenylalanine (DOPA) into proteins by replacing tyrosine with DOPA in the reaction mixtures. ESI-MS showed specific incorporation of DOPA in place of tyrosine. More than 90% DOPA incorporation at each tyrosine site was achieved, allowing the recording of clean N-15-HSQC NMR spectra. A redox-staining method specific for DOPA was shown to provide a sensitive and generally applicable method for assessing the cell-free production of proteins. Of four proteins produced in soluble form in the presence of tyrosine, two resulted in insoluble aggregates in the presence of high levels of DOPA. DOPA has been found in human proteins, often in association with various disease states that implicate protein aggregation and/or misfolding. Our results suggest that misfolded and aggregated proteins may result, in principle, from ribosome-mediated misincorporation of intracellular DOPA accumulated due to oxidative stress. High-yield cell-free protein expression systems are uniquely suited to obtain rapid information on solubility and aggregation of nascent polypeptide chains.

Antibacterial activity of bovine lactoferrin-derived peptides

Several peptides sharing high sequence homology with lactoferricin B (Lf-cin B) were generated from bovine lactoferrin (Lf) with recombinant chymosin. Two peptides were copurified. one identical to Lf-cin B and another differing from Lf-cin B by the inclusion of a C-terminal alanine (lactoferricin). Two other peptides were copurified from chymosin-hydrolyzed Lf. one differing from Lf-cin B by the inclusion of C-terminal alanyl-leucine and the other being a heterodimer linked by a disulfide bond, These peptides were isolated in a single step from chymosin-hydrolyzed Lf by membrane ton-exchange chromatography and were purified by reverse-phase high-pressure liquid chromatography (HPLC), They were characterized by. N-terminal Edman sequencing, mass spectrometry, and antibacterial activity determination, Pure lactoferricin, prepared from pepsin-hydrolyzed Lf, was purified by standard chromatography techniques, This peptide was analyzed against a number of gram-positive and gram-negative bacteria before and after reduction of its disulfide bond or cleavage after its single methionine residue and was found to inhibit the growth of all the test bacteria at a concentration of 8 mu M or less, Subfragments of lactoferricin were isolated from reduced and cleaved peptide by reverse-phase HPLC, Subfragment 1 (residues I to 10) was active against most of the test microorganisms at concentrations of 10 to 50 mu M. Subfragment 2 (residues 11 to 26) was active against only a few microorganisms at concentrations up to 100 mu M. These antibacterial studies indicate that the activity of lactoferricin Is mainly, but not wholly, due to its N-terminal region.

A family of cytokine-inducible inhibitors of signalling

Cytokines are secreted proteins that regulate important cellular responses such as proliferation and differentiation(1). Key events in cytokine signal transduction are well defined: cytokines induce receptor aggregation, leading to activation of members of the JAK family of cytoplasmic tyrosine kinases. In turn, members af the STAT family of transcription factors are phosphorylated, dimerize and increase the transcription of genes with STAT recognition sites in their promoters(1-4). Less is known of how cytokine signal transduction is switched off. We have cloned a complementary DNA encoding a protein SOCS-1, containing an SH2-domain, by its ability to inhibit the macrophage differentiation of M1 cells in response to interleukin-6. Expression of SOCS-1 inhibited both interleukin-6-induced receptor phosphorylation and STAT activation. We have also cloned two-relatives of SOCS-1, named SOCS-2 and SOCS-3, which together with the previously described CIS (ref. 5) form a new family of proteins. Transcription of all four SOCS genes is increased rapidly in response to interleukin-6, in vitro and in vivo, suggesting they may act in a classic negative feedback loop to regulate cytokine signal transduction.

Local dispersion of the Eucalyptus leaf beetle Chrysophtharta bimaculata (Coleoptera: Chrysomelidae), and implications for forest protection

1. Chrysophtharta bimaculata is a native chrysomelid species that can cause chronic defoliation of plantation and regrowth Eucalyptus forests in Tasmania, Australia. Knowledge of the dispersion pattern of C. bimaculata was needed in order to assess the efficiency of an integrated pest management (IPM) programme currently used for its control. 2. Using data from yellow flight traps, local populations of C. bimaculata adults were monitored over a season at spatial scales relevant to commercial forestry: within a 50-ha operational management unit (a forestry 'coupe') and between coupes. In addition, oviposition was monitored over a season at a subset of the between-coupe sites. 3. Dispersion indices (Taylor's Power Law and Iwao's Mean Crowding regression method) demonstrated that C. bimaculata adults were spatially aggregated within and between coupes, although the number of egg-batches laid at the between-coupe scale was uniform. Spatial autocorrelation analysis showed that trap-catches at the within-coupe level were similar (positively autocorrelated) to a radius distance of approximately 110 m, and then dissimilar (negatively autocorrelated) at approximately 250 m. At the between-coupe scale, no repeatable spatial autocorrelation patterns were observed. 4. For any individual site, rapid changes in beetle density were observed to be associated with loosely aggregated flights of beetles into and out of that site. Peak adult catches (> the weekly mean plus standard deviation trap-catch) for a site occurred for a period of 2.0 +/- 0.22 weeks at a time (n = 37), with normally only one or two peaks per site per season. Peak oviposition events for a site occurred on average 1.4 +/- 0.11 times per season and lasted 1.5 +/- 0.12 weeks. 5. Analysis of an extensive data set (n = 417) demonstrated that adult abundance at a site was positively correlated with egg density, but negatively correlated with tree damage (caused by conspecifics) and the presence of conspecific larvae. There was no relationship between adult abundance and a visual estimate of the amount of young foliage on trees. 6. Adults of C. bimaculata are show n to occur in relatively small, mobile aggregations. This means that pest surveys must be both regular (less than 2 weeks apart) and intensive (with sampling points no more than 150 m apart) if beetle populations are to be monitored with confidence. Further refinement of the current IPM strategy must recognize the problems posed by this temporal and spatial patchiness, particularly with regard to the use of biological insecticides, such as Bacillus thuringiensis, for which only a very short operational window exists.

The effect of the aged garlic extract, 'Kyolic', on the development of experimental atherosclerosis

The aged garlic extract 'Kyolic' lowers serum cholesterol levels in humans and experimental animals and thus is presumed to have a protective effect against atherosclerosis. However, to date no studies have examined the effect of this substance on the actual development of the disease. In the present study, the right carotid artery of 24 rabbits was de-endothelialized by balloon catheterisation in order to produce a myointimal thickening. After 2 weeks the rabbits were randomly assigned to four groups: Group I received a standard diet; Group II received the standard diet supplemented with 800 mu 1/kg body weight/day 'Kyolic'; Group III received a 1% cholesterol supplemented standard diet; and Group IV received a 1% cholesterol supplemented standard diet plus 'Kyolic'. After 6 weeks, the cholesterol diet caused a 6-fold increase in serum cholesterol level (Group III; 6.4 +/- 0.6 mmol/1) compared to normal diet (Group I; 1.2 +/- 0.4 mmol/1) (P < 0.05) with only a minor, non-significant reduction seen by the addition of 'Kyolic' (Group IV; 6.2 +/- 0.7 mmol/l). Group III rabbits developed fatty streak lesions covering approximately 70 +/- 8% of the surface area of the thoracic aorta, which was significantly reduced to 25 +/- 3% in the 'Kyolic'-treated Group IV. No lesions were present in Groups I and II. The hypercholesterolaemic diet caused an increase in aortic arch cholesterol (2.1 +/- 0.1 mg cholesterol/g tissue) which was significantly reduced by 'Kyolic' supplementation (1.7 +/- 0.2 mg cholesterol/g tissue) (P < 0.05). 'Kyolic' significantly inhibited the development of thickened, lipid-filled lesions in the pre-formed neointimas produced by balloon-catheter injury of the right carotid artery in cholesterol-fed rabbits (intima as percent of artery wall, Group III 42.6 +/- 6.5% versus Group IV 23.8 +/- 2.3%, P < 0.01), but had little effect in rabbits on a standard diet (Group II 18.4 +/- 5.0% versus Group I 16.7 +/- 2.0%). In vitro studies showed that 'Kyolic' has a direct effect on inhibition of smooth muscle proliferation. In conclusion,'Kyolic' treatment reduces fatty streak development, vessel wall cholesterol accumulation and the development of fibro fatty plaques in neointimas of cholesterol-fed rabbits, thus providing protection against the onset of atherosclerosis. (C) 1997 Elsevier Science Ireland Ltd.

Design analysis for refolding monomeric protein

Renaturation of protein expressed as inclusion bodies within Escherichia coli is a key step in many bioprocesses. Operating conditions for the refolding step dramatically affect the amount of protein product recovered, and hence profoundly influence the process economics. The first systematic comparison of refolding conducted in batch, fed-batch and continuous stirred-tank reactors is provided Refolding is modeled as kinetic competition between first-order refolding (equilibrium reaction) and irreversible aggregation (second-order). Simulations presented allow direct comparison between different flowsheets and refolding schemes using a dimensionless economic objective. As expected from examination of the reaction kinetics, batch operation is the most inefficient merle. For the base process considered, the overall cost of fed-batch and continuous refolding is virtually identical (less than half that of the batch process). Reactor selection and optimization of refolding using overall economics are demonstrated to be vitally important.

Slaking, dispersion, and crust formation

Soil structure is generally defined as the arrangement, orientation, and organization of the primary particles of sand, silt, and clay into compound aggregates, which exhibit properties that are unequal to the properties of a mass of nonaggregated material with a similar texture.6 Therefore the nature of soil structure is that it conveys specific properties to the soil and any alteration, i.e., breakdown or structural development, to the soil structural units will affect the physical properties of the soil. The aggregation and organization of the soil particles tend to form a hierarchical order4, 5 where the lower orders tend to have higher densities and greater internal strength than the higher orders. A schematic diagram of the hierarchical nature of soil structural elements in a clay soil is given in Fig. 1.4 Clay particles tend to form domains (packets of parallel clay sheets, generally consisting of 5-7 sheets), in turn several domains form clusters, followed by several orders of clusters, micro- and macroaggregates. The hierarchical nature implies that the destruction of a lower order will result in the destruction of all higher hierarchical orders. An example is the dispersion of sodic clay domains which results in the destruction of all higher orders, resulting in a dense soil with low hydraulic conductivity. Hence the clay domains are the fundamental building blocks of the soil and its integrity may determine the soil's physical properties and behavior.

Emotions at multiple levels: An integration

Weiss and Isen have provided many supportive comments about the multi-level perspective, but also found limitations. Isen noted the importance of integrating affect, cognition, and motivation. Weiss commented similarly that the model lacked an integrating “thread.” He suggested that, to be truly multilevel, each level should constrain processes at other levels, and also provide guidance for the development of new concepts. Weiss also noted that the focus on biological processes was a strength of the model. I respond by suggesting that these very biological processes may constitute the “missing” thread. To illustrate this, I discuss some of the recent research on emotions in organizational settings, and argue that biology both constrains and guides theory at each level of the model. Based on this proposition, I revisit each of the five levels in the model, to demonstrate how this integration can be accomplished in this fashion. Finally, I address two additional points: aggregation bias, and the possibility of extending the model to include higher levels of industry and region.

Towards a molecular epidemiology of alcohol dependence analysing the interplay of genetic and environmental risk factors

Background Progress in identifying genetic factors protective against alcohol dependence (AlcD) requires a paradigm shift in psychiatric epidemiology. Aims To integrate analysis of research into the genetics of alcoholism. Method Data from prospective questionnaire and interview surveys of the Australian twin panel, and from a subsample who underwent alcohol challenge, were analysed. Results In men, effects of alcohol dehydrogenase ADH2*1/*2 genotype or high alcohol sensitivity (risk-decreasing), and of history of childhood conduct disorder, or having monozygotic co-twin or twin sister with AlcD (risk-increasing) were significant and comparable in magnitude. Religious affiliation (Anglican versus other) was associated with the ADH2 genotype, but did not explain the associations with AlcD symptoms. No protective effect of the ADH2*1/*2 genotype was observed in women. Conclusions The early onset and strong familial aggregation of AlcD, and opportunity for within-family tests of genetic association to avoid confounding effects, make epidemiological family studies of adolescents and young adults and their families a priority.

The genetic and environmental relationship between the interpersonal sensitivity measure (IPSM) and the personality dimensions of Eysenck and Cloninger

A shortened version of the Interpersonal Sensitivity Measure (IPSM) developed to predict depression prone personalities was administered in a self-report questionnaire to a community-based sample of 3269 Australian twin pairs aged 18-28 years, along with Eysenck's EPQ and Cloninger's TPQ. The IPSM included four sub-scales: Separation Anxiety (SEP); Interpersonal Sensitivity (INT); Fragile Inner-Self (FIS); and Timidity (TIM). Univariate analysis revealed that individual differences in the IPSM sub-scale scores were best explained by additive genetic and specific environmental effects. Confirming previous research findings, familial aggregation for the EPQ and TPQ personality dimensions was entirely due to additive genetic effects. In the multivariate case, a model comprising additive genetic and specific environmental effects best explained the covariation between the latent factors for male and female twin pairs alike. The EPQ and TPQ dimensions accounted for moderate to large proportions of the genetic variance (40-76%) in the IPSM sub-scales, while most of the non-shared environment variance was unique to the IPSM sub-scales. (C) 2001 Elsevier Science Ltd. All rights reserved.

A new wavelet-based method for the solution of the population balance equation

A new wavelet-based method for solving population balance equations with simultaneous nucleation, growth and agglomeration is proposed, which uses wavelets to express the functions. The technique is very general, powerful and overcomes the crucial problems of numerical diffusion and stability that often characterize previous techniques in this area. It is also applicable to an arbitrary grid to control resolution and computational efficiency. The proposed technique has been tested for pure agglomeration, simultaneous nucleation and growth, and simultaneous growth and agglomeration. In all cases, the predicted and analytical particle size distributions are in excellent agreement. The presence of moving sharp fronts can be addressed without the prior investigation of the characteristics of the processes. (C) 2001 Published by Elsevier Science Ltd.