Use of virus-like particles as a native membrane model to study the interaction of insulin with the insulin receptor

There is emerging evidence of the utility of virus-like particles (VLPs) as a novel model for the study of receptor-ligand interactions in a native plasma membrane environment. VLPs consist of a viral core protein encapsulated by portions of the cell membrane with membrane proteins and receptors expressed in their native conformation. VLPs can be generated in mammalian cells by transfection with the retroviral core protein (gag). In this study, we used Chinese hamster ovary (CHO T10) cells stably overexpressing the insulin receptor (IR) to generate IR bearing VLPs. The diameter and size uniformity of VLPs were estimated by dynamic light scattering and morphological features examined by scanning electron microscopy. The presence of high affinity IR on VLPs was demonstrated by competitive binding assays (K_D: 2.3 ± 0.4 nM, n = 3), which was similar to that on the parental CHO T10 cells (K_D: 2.1 ± 0.4 nM, n = 3). We also report that increases or decreases in membrane cholesterol content by treatment with methyl-β-cyclodextrin (MBCD) or cholesterol pre-loaded methyl-β-cyclodextrin (cMBCD), respectively, substantially decreased insulin binding (> 30%) to both VLPs and cells, and we speculate this is due to a change in receptor disposition. We suggest that this novel finding of decreases in insulin binding in response to changes in membrane cholesterol content may largely account for the unexplained decreases in insulin signalling events previously reported elsewhere. Finally, we propose VLPs as a viable membrane model for the study of insulin-IR interactions in a native membrane environment.     

A method to evaluate the effect of liposome lipid composition on its interaction with the erythrocyte plasma membrane

Lipid aggregates are considered promising carriers for macromolecules and toxic drugs. In order to fulfill this function, aggregates should have properties that ensure the efficient delivery of their cargo to the desired location. One of these properties is their stability in blood when accumulating in the targeted tissue. This stability may be affected by a number of factors, including enzymatic activity, protein adsorption, and non-specific lipid exchange between the aggregate and morphological blood components. Since blood cells in the majority consist of erythrocytes, their interaction with aggregates should be carefully analyzed. In this paper, we present a method that allows the exchange of lipid between liposomes and the erythrocyte plasma membrane to be evaluated. The extent of this exchange was measured in terms of the toxicity of a cationic lipid (DOTAP) incorporated into the liposome lipid bilayer, evaluated by plasma membrane mechanical properties. After liposomes were formed from DOTAP/PC or DOTAP/PE mixtures, erythrocyte plasma membranes were destabilized in a manner dependent on DOTAP concentration. A constant quantity of DOTAP mixed with various proportions of SM caused no such effect, indicating very limited lipid exchange with the cell membrane for such liposome formulations.     

Dietary fat,fatty acid composition in plasma and the metabolic syndrome

PURPOSE OF REVIEW: The metabolic syndrome, a cluster of disorders often including abdominal obesity, is associated with a high risk of cardiovascular disease and premature death. Insulin resistance is a key feature of the metabolic syndrome. Observational studies have indicated that the type of fat in the diet may be related to the development of insulin resistance and the metabolic syndrome, also independent of possible effects on body weight. Dietary surveys are often imprecise. One way to monitor the type of fat in the diet is to record the fatty acid composition in plasma. This review summarizes recent data on the relationships between fatty acid composition in plasma and insulin resistance, diabetes and other disorders related to the metabolic syndrome. RECENT FINDINGS: Insulin resistance and insulin resistant states are often associated with the fatty acid pattern in plasma, characterized by an increased proportion of palmitic (16 : 0) and a low proportion of linoleic (18 : 2 n-6) acids, with a distribution of other fatty acids indicating an increased activity of delta-9 and delta-6 desaturase. This shows that there may be a causal relationship between the type of fat in the diet and insulin action, an assumption supported by recent dietary intervention studies. SUMMARY: In a public health perspective these results, from both observational and intervention studies, underline the importance of fat quality in the diet for the development of a number of prevalent diseases. Taken together with several earlier studies and recent epidemiological findings, they give strong support to present dietary guidelines.     

Use of circular dichroism to study the interaction of carboxypeptidase A with substrates and inhibitors

The phenylalanyl circular dichroism (CD) bands of peptides were used to assay peptidase activity of carboxypeptidase A (EC.3.4.12.2.). Hippuryl-L-phenylalanine has a sharp, negative CD band at 254 nm whilst L-phenylalanine (the optically active product) has positive CD. Thus the hydrolysis of this substrate as well as the inhibition effect of dipeptides, may be measured from the CD change at 254 nm. The addition of the dipeptide GLy-Tyr to carboxypeptidase A makes the CD spectrum more positive in the region from 270-295 nm. This alteration can result from the tyrosyl and tryptophanyl CD bands of the protein as well as from the tyrosyl CD band of the inhibitor.     

Use of the fluorescent probe method to study the interaction of barbiturates with biological membranes]

The binding of the negatively charged fluorescence dye ANS and neutral dye NPN2 with lipid and erythrocyte membranes in the presence of barbiturates was studied. It was found that barbiturates decreased the amount of binding sites of ANS and NPN2 with membranes did not affect the quantum yield and the dissociation of the membrane-dye complex. It was shown that all barbiturates investigated were bound with the membranes in a neutral form.     

Use of small angle neutron scattering to study the interaction of angiotensin II with model membranes

Understanding biological processes assumes a detailed understanding of the interaction of all involved molecules. Here the effect of the peptide hormone angiotensin II (Ang II), an agonist of the angiotensin receptors, on the structure of unilamellar and multilamellar dimyristoyl phosphatidylcholine vesicles was studied by small angle neutron scattering, dynamic light scattering and differential scanning calorimetry. The calorimetry data indicate a weak interaction of Ang II with the surface of the membrane bilayer, as the pretransition persists during all experiments, and the main transition is only slightly shifted towards higher temperatures. From the SANS data we were able to confirm the calorimetric data and verify the interaction of the hormone with the membrane surface. At low temperatures, when the lipid molecules are in the gel phase, more precisely in the ripple phase, the peptide penetrates in the head group core, but due to the close packing of the acyl chains, the hydrophobic region is not affected. In a temperature region below but close to the region of the phase transition, the hydrophibic core starts to be affected by the peptide, and the same is true for the fluid phase. Upon binding of the peptide, the thickness of the head group increases, and the scattering length density of the head group starts to rise with increasing peptide concentrations. This interaction and binding to the membrane surface may be relevant for the relocation, binding and reconstitution of the angiotensin receptors into the membrane. Second, the peptide adsorption to the membrane surface may contribute to the binding of Ang II in the active site of the receptor.     

Use of resistant mutants to study the interaction of triton X-100 with Staphylococcus aureus.

Staphylococcus aureus mutants resistant to the nonionic detergent Triton X-100, isolated from the wild-type strain H and the autolysin-deficient strain RUS3, could grow and divide in broth containing 5% (vol/vol) Triton X-100, while growth of the parental strains was markedly inhibited above the critical micellar concentration (0.02%) of the detergent. Growth-inhibitory concentrations of Triton X-100 killed wild-type cells without demonstrable cellular lysis. Triton X-100 stimulated autolysin activity of S. aureus cells under nongrowing conditions, and this lytic response was markedly reduced in energy-poisoned cells. In contrast, the detergent had no effect on the activity of autolysins in cell-free systems, and growth in the presence of Triton X-100 did not alter either the cellular autolysin activity or the susceptibility of cell walls to exogenous lytic enzymes. Treatment with either Triton X-100 or penicillin G in the growth medium stimulated release of predominantly acylated intracellular lipoteichoic acid and sensitized staphylococci to Triton X-100-induced autolysis. There was no significant difference in the cell wall and membrane compositions or Triton X-100 binding between the parental strains and the resistant mutants. The resistant mutant TXR1, derived from S. aureus H, had a higher level of L-alpha-glycerophosphate dehydrogenase activity, and its oxygen uptake was more resistant to inhibition by a submicellar concentration (0.008%) of Triton X-100. Growth in the presence of subinhibitory concentrations of Triton X-100 rendered S. aureus H cells phenotypically resistant to the detergent and greatly stimulated the level of oxygen uptake. Membranes isolated from such cells exhibited enhanced activity of the respiratory enzymes succinic dehydrogenase and L-alpha-glycerophosphate dehydrogenase.     

Use of cold carcass weight and fat depth measurements to predict carcass composition of Rasa Aragonesa lambs

A total of 22 measurements of fat depth, taken along the 13th rib, 5–6th lumbar vertebra, 3–4th sacrum vertebra, and the 2nd, 2–3rd, 3rd, 3–4th, 4th, 4–5th sternebra of the breast bone, were taken on intact carcasses with a sharpened steel rule, and related to the carcass composition of 18 Rasa Aragonesa lambs. The objective was to study the accuracy of these different measurements for predicting carcass composition and possible value, in the process of carcass grading or classification of ‘Ternasco’ Aragón lambs. Cold carcass weight (CCW) accounted for 74% and 40% of the total variation of muscle weight and total carcass fat weight, respectively. The addition of 4 cm fat depth over the 13th rib from the left side accounted for a further 13% of muscle weight, and the addition of 4 cm fat depth over the 5–6th lumbar vertebra from the right side accounted for a further 29% of the variation of total carcass fat weight. Regarding different fat depots, CCW accounted for 49% and 31% of the total variation of the intermuscular fat and kidney and pelvic fat, respectively.

However, CCW alone only accounted for 21% (NS) of the total variation of subcutaneous fat weight, but the addition of 4 cm fat depth over the 13th rib from the right side or the addition of 4 cm fat depth over the 5–6th lumbar vertebra from the right side accounted for a further 47% of the subcutaneous fat variation. A comparison of the residual standard deviations (RSD) of the two measurements indicated that the fat depth over the 5–6th lumbar vertebra was more accurate for predicting subcutaneous fat. The highest precision, after CCW, for predicting intermuscular fat, was fat depth over the 13th rib from the left side, which accounted for a further 18% of total variation of this fat depot. Prediction of carcass composition was improved by the addition of fat depth measurements, assisting in the commercial classification of ‘Ternasco’ Aragón lambs.     

Use of fluorescence enhancement technique to study bilirubin–albumin interaction

Bilirubin–albumin solution gave an emission spectrum in the wavelength range 500–600 nm with emission maxima at 528 nm when excited at 487 nm. The magnitude of fluorescence intensity increased on increasing bilirubin/albumin molar ratio. At three different albumin concentrations, namely, 1.0, 2.5 and 10.0 μM, there was an initial linear increase in fluorescence up to a molar ratio 1.0 in all cases beyond which it sloped off or decreased. This fluorescence enhancement was used to calculate the binding parameters of bilirubin–albumin interaction and the value of binding constant was found to be 1.72×10⁷ l/mol similar to the published values obtained with other methods. Different serum albumins, namely, human (HSA), goat (GSA), pig (PSA) and dog serum albumins (DSA) bound bilirubin with almost the same affinity when studied by the technique of fluorescence enhancement. Bilirubin–albumin interaction was also studied at different pH and ionic strengths. There was a decrease in bilirubin–albumin complex formation on either decreasing the pH from 9.0 to 7.0 or increasing the ionic strength from 0.15 to 1.0. These results suggest that the technique of fluorescence enhancement can be used successfully to study the bilirubin–albumin interaction.     

Use of primary fat body cultures for the study of baculovirus replication

Fat body cultures of Trichoplusia ni and Estigmene acrea were established for use in the study of the two baculoviruses Autographa californica nuclear polyhedrosis virus (AcNPV) and Estigmene acrea granulosis virus (EaGV), respectively. Multiplication of AcNPV observed by phase and electron microscopy was correlated with an increase in viral specific proteins as determined by indirect enzyme-linked immunosorbent assay (ELISA). Although EaGV morphogenesis was not observed in fat body cultures, an increase in specific proteins of this virus could be detected with the ELISA.     

Dietary fat composition modifies the effect of boron on bone characteristics and plasma lipids in rats

Female and male rats weighing about 170 g and 200 g, respectively, were fed diets (approximately 70 microg boron/kg) in a factorial arrangement with supplemental boron at 0 (deficient) and 3 (adequate) mg/kg and canola oil or palm oil at 75 g/kg of diet as variables. After 5 weeks, six females in each treatment were bred. Dams and pups continued on their respective dietary treatments through gestation, lactation and post-weaning. Thirteen weeks after weaning, plasma and bones were collected from 12 male and 12 female offspring in each treatment. Boron supplementation increased femur strength measured by the breaking variable bending moment; tibial calcium and phosphorus concentrations; and plasma alkaline phosphatase. Femur breaking stress was greatest in boron-supplemented rats fed canola oil, and lowest in boron-deprived females fed canola oil; this group also exhibited the lowest femur bending moment. Minerals associated with bone organic matrix, zinc and potassium, were increased by boron supplementation in tibia. Plasma phospholipids were decreased by boron deprivation in females, but not males. Plasma cholesterol was decreased in boron-supplemented males by replacing canola oil with palm oil. The findings suggest that a diet high in omega-3 alpha-linolenic acid promotes femur strength best when the dietary boron is adequate.     

Use of transgenic plants and mutants to study the regulation and function of lipid composition

Mutants and transgenic plants with altered expression of genes implicated in lipid metabolism are providing fresh insights into the regulation and function of lipid composition. To date, several genes encoding fatty acid desaturases, acyltransferases, a thioesterase, a lipid transfer protein and an isoform of acyl-carrier protein have been introduced into transgenic plants. Despite the fact that some of these transgenic plants had large alterations in lipid composition, they showed surprisingly little phenotypic variation from wild-type plants. Although detailed analyses of these plants are just beginning, several theories regarding the roles of particular genes in various plant processes, such as cold tolerance and transfer of lipids between membranes, have been either substantiated or discarded on the basis of the data already obtained. In addition, constructs that contain the promoter regions of genes implicated in lipid metabolism fused to reporter genes have been introduced into transgenic plants and are providing some clues as to how lipid composition is regulated.     

Design of bio-mimetic particles with enhanced vascular interaction

The majority of particle-based delivery systems for the ‘smart’ administration of therapeutic and imaging agents have a spherical shape, are made by polymeric or lipid materials, have a size in the order of few hundreds of nanometers and a negligibly small relative density to aqueous solutions. In the microcirculation and deep airways of the lungs, where the creeping flow assumption holds, such small spheres move by following the flow stream lines and are not affected by external volume force fields. A delivery system should be designed to drift across the stream lines and interact repeatedly with the vessel walls, so that vascular interaction could be enhanced. The numerical approach presented in [Gavze, E., Shapiro, M., 1997. Particles in a shear flow near a solid wall: effect of nonsphericity on forces and velocities. International Journal of Multiphase Flow 23, 155–182.] is, here, proposed as a tool to analyze the dynamics of arbitrarily shaped particles in a creeping flow, and has been extended to include the contribution of external force fields. As an example, ellipsoidal particles with aspect ratio 0.5 are considered. In the absence of external volume forces, a net lateral drift (margination) of the particles has been observed for Stokes number larger than unity (St>1); whereas, for smaller St, the particles oscillate with no net lateral motion. Under these conditions, margination is governed solely by particle inertia (geometry and particle-to-fluid density ratio). In the presence of volume forces, even for fairly small St, margination is observed but in a direction dictated by the external force field. It is concluded that a fine balance between size, shape and density can lead to EVI particles (particles with enhanced vascular interaction) that are able to sense endothelial cells for biological and biophysical abnormalities, mimicking circulating platelets and leukocytes.     

Use of ion exchange chromatography for the study of RecA-DNA interaction

In vitro binding of RecA protein to double-stranded DNA (dsDNA) was studied using ion-exchange liquid chromatography. The method allowed quantification of both free DNA and free protein. The results unambiguously showed a binding stoichiometry of 3 base pairs per RecA monomer. The binding exhibited cooperativity, and the stoichiometry suggested that RecA does not form complexes with two molecules of dsDNA. More than 90% of RecA molecules in the sample were active for DNA binding.     

Probing the interaction of arsenobetaine with blood plasma constituents in vitro: an SEC-ICP-AES study

Arsenobetaine, which is frequently ingested by humans via the consumption of seafood, is rapidly excreted unchanged in urine, but not much is known about its transport in the mammalian bloodstream. To assess whether this transport involves binding to plasma proteins, rabbit and human plasma were spiked with arsenobetaine and the mixture was analyzed (after 5 min and again after 6 h) by size-exclusion chromatography (SEC) coupled on-line to an inductively coupled plasma atomic emission spectrometer (ICP-AES). Simultaneous monitoring of the emission lines of As, Cu, Fe and Zn in the column effluent allowed us to determine the elution of arsenobetaine relative to that of the major Cu, Fe and Zn-containing metalloproteins. Over the investigated time period, a single As peak eluted near the inclusion volume on two different SEC columns with fractionation ranges of 600-10 KDa and 7000-100 Da. These results indicate that arsenobetaine did not bind to plasma proteins and that SEC-ICP-AES is a useful tool to rapidly probe toxicologically and pharmacologically-relevant interactions between organometalloid compounds and mammalian blood plasma constituents in vitro.