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PubMed1995

A new methodology for non-invasive clinical assessment of cardiovascular system performance and of ventricular-arterial coupling during stress.

The objective of the study was to develop a non-invasive method for the quantitative evaluation of cardiovascular performance and ventricular-arterial (VA) coupling during varying physiological states. VA-coupling was represented by the ratio between the arterial and ventricular elastances-Ea/Ees. Approximate indices of the relative change of Ees and VA-coupling during stress were developed and tested. These indices can be evaluated directly from non-invasive measurements of ejection fraction values (for VA-coupling) and measurements of stroke volumes and systolic and diastolic arterial pressures (for Ees). Additional relative indices can be evaluated from these data (e.g., stroke work, cardiac output) to yield a complete representation of the cardiovascular response to stress. The present methodology was applied to assess the exercise stress response in healthy subjects (H, n = 8) and in patients with left ventricular dysfunction (n = 24). Left ventricular volumes were determined by nuclear angiography and arterial pressures were measured non-invasively by a new, validated method. Using published data obtained invasively, we found that the relative indices of Ees and VA-coupling showed a high correlation with the invasive ones (r > 0.8, P < 0.01). The patients were subgrouped by their maximal exercise capacitance (P2-50W, P3-75W). At rest, the two patient groups had similar ejection fraction values (45 +/- 15% and 48 +/- 16%), which were significantly different from those of the healthy subjects (66 +/- 7%, P < 0.05). During stress, a larger increase in stroke work and cardiac output was found in the healthy subjects. All three groups showed similar relative increases in Ees and heart rate, but relative Ea increased in P2 and decreased in H, while the opposite was found for the end-diastolic volume. The relative VA-coupling index in P2 was significantly larger than that in P3 and H (P < 0.05). The present non-invasively based indices can be used to quantitatively monitor the individual cardiovascular response to stress testing or drug interventions and to evaluate the importance of VA-coupling in the clinical setting.

Arteries↗

PubMed1982

Energy coupling to ATP synthesis by the proton-translocating ATPase.

This review summarizes recent work on energy coupling to ATP synthesis by the reversible, proton-translocating ATPase to mitochondria, chloroplasts, and bacteria. In the first sections, this enzyme is distinguished from other ATP-linked ion transport systems, and progress in the biochemical analysis is discussed. There is at present a reasonably consistent idea of the overall structure of the enzyme, and one can begin to assign specific functional roles to individual subunits of the complex. The latter half of the review deals with mechanisms of energy coupling, about which there is clear divergence of opinion. An "indirect coupling" model would allow for the possibility that H+ translocation transmits energy for ATP synthesis by driving the enzyme through a sequence of conformational states, so that H+ translocated need not take part in the chemistry of ATP synthesis. By contrast, a "direct coupling" mechanism would specify that H+ translocated must participate in the chemical reaction by combining with oxygen must participate in the chemical reaction by combining with oxygen from phosphate during the synthetic step. Such discussion is preceded by an outlined of the "proton well," since this idea forms the basis of one direct coupling model. In addition, it is suggested that the idea of a proton (ion) well may be of more general significance to the analysis of ion-coupled transport, because it includes the postulate that mechanistically significant ion binding can occur within the profile of the electric field. A proton (ion) well can be derived from both kinetic and equilibrium treatments, and from mechanistic considerations in fields as distinct as biochemistry and neurophysiology. As a result, it illustrates how further advances in formulating mechanisms of energy coupling might profit by a merger of technique and perspective from areas that have as a common goal an understanding of how large proteins catalyze movements of small molecules across a membrane.

Adenosine Triphosphatases↗

PubMed1998

Similar effects of cis-diamminedichloroplatinum(II) and cis-diammine-1,1-cyclobutanedicarboxylatoplatinum(II) on sodium-coupled glucose uptake in renal brush-border membrane vesicles.

cis-Diamminedichloroplatinum(II) (cDDP) has been shown to interfere with reabsorption processes in renal tubular epithelia, leading to polyuria, magnesium and sodium wasting and glucosuria. cDDP inhibits the Na+-coupled uptake of methyl-alpha-D-glucopyranoside (MGP) in renal proximal tubular cells in primary culture. cis-Diammine-1,1-cyclobutane dicarboxylatoplatinum(II) (CBDCA) produces tubular injury qualitatively similar to that of cDDP with a reduced severity. CBDCA inhibits Na+-coupled MGP uptake in renal proximal tubular cells in primary culture at concentrations 20- to 30-times higher than those of cDDP. The Na+/glucose cotransport protein possesses sulphydryl groups (SH) essential for its activity. Platinum complexes have strong affinity for SH groups. We compared the direct effects of cDDP (0.04-1.0 mM) and CBDCA (1-30 mM) on Na+-coupled MGP uptake in rabbit renal brush-border membrane (BBM) vesicles. cDDP and CBDCA inhibited Na+-coupled MGP uptake in a concentration-dependent manner, mainly through a decrease in Vmax of the cotransport protein. These effects were associated with platinum binding to BBM and decreases in protein-bound SH groups. CBDCA altered Na+-coupled MGP uptake at concentrations 30-times higher than those of cDDP. When BBM vesicles were preincubated with cDDP or CBDCA, diethyldithiocarbamate (an antidote against cDDP-induced nephrotoxicity) partly restored Na+-coupled MGP uptake and reduced the amount of platinum bound to BBM, but did not restore protein-bound SH groups. These findings strongly suggest that the inhibition of Na+-coupled MGP uptake by cDDP and CBDCA is mainly mediated by direct chemical binding of platinum to essential SH groups of the cotransport protein but may also involve other nucleophilic groups, such as the SCH3 group of methionine residues.

Animals↗

PubMed2003

Tachycardia exacerbates abnormal left ventricular-arterial coupling in heart failure.

The purpose of this study was to assess the effect of heart rate on left ventricular (LV)-arterial coupling and LV mechanical efficiency before and after heart failure (CHF). The production of LV stroke work (SW) and mechanical efficiency depends on the coupling of the LV and arterial system. The response of LV-arterial coupling to tachycardia may be altered in heart failure. We compared the response of LV-arterial coupling to increased heart rate (HR) in six conscious, instrumented dogs before and after pacing-induced CHF. Coupling was quantified as E(ES)/ E(A), where E(ES) is the slope of end-systolic pressure ( P)-volume ( V) relation, and E(A) is arterial elastance. Mechanical efficiency was determined as the ratio of SW to a total P-V area (PVA). Before CHF, E(ES) and E(A) increased similarly with increased heart rate to 180 min(-1). Thus, E(ES)/ E(A) remained unaltered (0.96 +/- 0.08 vs 0.94 +/- 0.35), and SW/PVA was unchanged (0.62 +/- 0.03 vs 0.59 +/- 0.06). Compared with the results prior to CHF and after CHF the resting E(ES) was decreased, thus both E(ES)/ E(A) (0.58 +/- 0.09) and SW/PVA (0.48 +/- 0.06) were less ( P << 0.05) than baseline. After CHF, an increase in HR to 180 min(-1) increased E(A) but not E(ES), thus E(ES)/ E(A) fell to 0.44 +/- 0.06 ( P << 0.05) and SW/PVA fell to 0.41 +/- 0.05 ( P << 0.05). Under normal conditions, LV-arterial coupling remains optimal during increases in HR. After CHF, tachycardia exacerbates the suboptimal baseline LV-arterial coupling, reducing the efficiency of producing SW.

Animals↗

PubMed1997

Parallel processing by a homogeneous group of coupled model neurons can enhance, reduce and generate signal correlations.

Correlated activities have been proposed as correlates of flexible association and assembly coding. We addressed the basic question of how signal correlations on parallel pathways are enhanced, reduced and generated by homogeneous groups of coupled neurons, and how this depends on the input activities and their interactions with internal coupling processes. For this we simulated a fully connected group of identical impulse-coded neurons with dynamic input and threshold processes and additive or multiplicative lateral coupling. Input signals were Gaussian white noise (GWN), completely independent or partially correlated on a subgroup of the parallel inputs. We show that in states of high average spike rates input-output correlations were weak while the network could generate correlated activities of stochastic, oscillatory and rhythmic bursting types depending exclusively on lateral coupling strength. In states of low average spike rates input-output correlations were high and the network could effectively enhance or reduce differences in spatial correlation applied to its parallel inputs. The correlation differences were more pronounced with multiplicative lateral coupling than with the additive interactions commonly used. As the different modes of correlation processing emerged already by global changes in the average spike rate and lateral coupling strength, we assume that in real cortical circuits changes in correlational processing may also be induced by unspecific modulations of activation and lateral coupling.

Action Potentials↗

PubMed2001

Non-linear coupling of atrial activation processes during atrial fibrillation in humans.

The activation patterns underlying the electrical activity of the heart during atrial fibrillation (AF) are not entirely random. The aim of this study was to assess the local organization of the activation processes during AF by estimating the non-linear coupling between activation sequences (ASs) in two atrial sites. To quantitatively estimate the degree of non-linear coupling we extracted two indices based on a multivariate embedding procedure and on the estimation of the correlation dimension (CD) and correlation entropy (CE), termed independence of complexity and of independence of predictability, respectively. We analysed AS in two atrial sites in 30 informed subjects during chronic AF of type I, II and III (Wells' classification), ten 6-s-long episodes of each type. Surrogates were used to reject the hypothesis that the time series were generated by linear stochastic dynamics. We estimated CD and CE according to the coarse-grained approach, which leads to a fixed high value for the embedding dimension in all the analysed ASs, and a typical value for the distance between the two ASs in the phase space. Various degrees of organization, ranging from completely synchronized to fully de-coupled signals, were observed: significant degrees of non-linear coupling were found in segments belonging in types I and II AF, whereas type III electrograms always turned out to be weakly coupled. This finding links the morphology of single electrograms to the synchronization between pairs of closely spaced electrograms. Our bivariate approach suggests that the measurement of organization during AF should be based on the estimation of the non-linear coupling between two sites. This approach appears to be more reliable and sensitive than non-linear analysis of single electrograms or linear analysis of their coupling.

Adult↗

PubMed2000

Intraprotoplasmic and wall-localised formation of arabinoxylan-bound diferulates and larger ferulate coupling-products in maize cell-suspension cultures.

Maize (Zea mays L.) cell cultures incorporated radioactivity from [14C]cinnamate into hydroxycinnamoyl-CoA derivatives and then into polysaccharide-bound feruloyl residues. Within 5-20 min, the CoA pool had lost its 14C by turnover and little or no further incorporation into polysaccharides then occurred. The system was thus effectively a pulse-chase experiment. Kinetics of radiolabelling of diferulates (also known as dehydrodiferulates) varied with culture age. In young (1-3 d) cultures, polysaccharide-bound [14C]feruloyl- and [14C]diferuloyl residues were both detectable within 1 min of [14C]cinnamate feeding. Thus, feruloyl residues were dimerised < 1 min after their attachment to polysaccharides. For at least the first 2.3 h after [14C]cinnamate feeding, polysaccharide-bound [14C]diferuloyl residues remained almost constant at approximately 7% of the total polysaccharide-bound [14C]ferulate derivatives. Since feruloyl residues are attached to polysaccharides < 1 min after the biosynthesis of the latter, and > 10 min before secretion, the data show that extensive feruloyl coupling occurred intra-protoplasmically. Exogenous H2O2 (1 mM) caused little additional feruloyl coupling; therefore, wall-localised coupling may have been peroxidase-limited. In older (e.g. 4 d) cultures, less intraprotoplasmic coupling occurred: during the first 2.5 h, polysaccharide-bound [14C]diferuloyl residues were a steady 1.4% of the total polysaccharide-bound [14C]ferulate derivatives. In contrast to the situation in younger cultures, exogenous H2O2 induced a rapid 4- to 6-fold increase in all coupling products, indicating that coupling in the walls was H2O2-limited. In both 2- and 4-d-old cultures, polysaccharide-bound 14C-trimers and larger coupling products exceeded [14C]diferulates 3- to 4-fold, but followed similar kinetics. Thus, although all known dimers of ferulate can now be individually quantified, it appears to be trimers and larger products that make the major contribution to cross-linking of wall polysaccharides in cultured maize cells. We argue that feruloyl arabinoxylans that are cross-linked before and after secretion are likely to loosen and tighten the cell wall, respectively. The consequences for the control of cell expansion and for the response of cell walls to an oxidative burst are discussed.

Acyl Coenzyme A↗

PubMed2005

Communication and frightening behavior among couples with past and recent histories of physical marital aggression.

The present study compares observed communication and couples' reports of husbands' frightening behaviors among couples with no history of husbands' physical marital aggression, a past history of such aggression, and recent history of such aggression. Ninety community couples reported on husbands' physical marital aggression during two visits to the lab, approximately 1.5 years apart. During the second visit, we videotaped couples' discussions of conflictual marital issues. Poor communication, as indexed by high hostility, low problem-description and low warmth, characterized couples with recent aggression, as compared to past aggression or no aggression. Husbands' frightening behavior characterized the couples with either recent or past aggression, compared to no aggression. In addition, for couples who had experienced severe aggression, the rates of husbands' frightening behavior were similar regardless of whether husbands persisted, reduced or desisted in the severe aggression. Discussion addresses possible reasons that reductions and desistance in husbands' aggression may be associated with more positive communication patterns but with continued reports of frightening behavior.

Adult↗

PubMed2005

Efficient and precise measurement of H(alpha)-C(alpha), C(alpha)-C', C(alpha)-C(beta) and H(N)-N residual dipolar couplings from 2D H(N)-N correlation spectra.

A suite of experiments are presented for the measurement of H(alpha)-C(alpha), C(alpha)-C', C(alpha)-C(beta) and H(N)-N couplings from uniformly 15N, 13C labeled proteins. Couplings are obtained from a series of intensity modulated two-dimensional H(N)-N spectra equivalent to the common 1H-15N-HSQC spectra, alleviating many overlap and assignment issues associated with other techniques. To illustrate the efficiency of this method, H(alpha)-C(alpha), C(alpha)-C', and H(N)-N isotropic scalar couplings were determined for ubiquitin from data collected in less than 4.5 h, C(alpha)-C(beta) data collection required 10 h. The resulting couplings were measured with an average error of +/-0.06, +/-0.05, +/-0.04 and +/-0.10 Hz, respectively. This study also shows H(alpha)-C(alpha) and C(alpha)-C(beta) couplings, valuable because they provide orientation of bond vectors outside the peptide plane, can be measured in a uniform and precise way. Superior accuracy and precision to existing 3D measurements for C(alpha)-C' couplings and increased precision compared to IPAP measurements for H(N)-N couplings are demonstrated. Minor modifications allow for acquisition of modulated H(N)-C' 2D spectra, which can yield additional well resolved peaks and significantly increase the number of measured RDCs for proteins with crowded 1H-15N resonances.

Carbon↗

PubMed1983

Coupling interval and types of ventricular ectopic activity associated with ventricular runs.

Repetitive 10-hour ECG recordings of 289 patients obtained within a year after myocardial infarction were analyzed for the presence of ventricular runs, and their association with various types of ventricular ectopic activity (VEA), average rate of premature ventricular complexes (PVCs), and coupling interval of PVCs initiating the runs. Tapes which contained complex VEA (bigeminy, multiform PVCs, couplets, or runs), early PVCs (coupling interval less than 400 msec), or late PVCs (coupling interval greater than 600 msec) had a substantially higher average PVC rate than those without them. The occurrence of ventricular runs was significantly more likely in tapes having other complex VEA or late PVCs than in those without them, and when tapes were stratified by PVC rates the presence of these dysrhythmias appeared to have an independent predictive value for the occurrence of runs. In contrast, the influence of early PVCs on the occurrence of runs was rather minimal, and this seemed to be related to their common association with the PVC rate. Furthermore, a larger percentage of isolated PVCs had coupling intervals between 400 msec and 600 msec, and most couplets (77%) and runs (67.8%) were initiated by PVCs with coupling intervals in this range. However, the proportion of either short (less than 400 msec) or long (greater than 600 msec) coupling interval PVCs initiating couplets or runs was significantly higher than those with intermediate coupling intervals (between 400 and 600 msec).

Arrhythmias, Cardiac↗

PubMed1988

Coupling intervals of ventricular extrastimuli causing resetting of sustained ventricular tachycardia secondary to coronary artery disease: relation to subsequent termination.

Single and double ventricular extrastimuli (VE) delivered during sustained, uniform ventricular tachycardia (VT) are able to reset or terminate the tachycardia. The relation between the coupling intervals of single and double VE resetting VT and those terminating it was examined in 80 uniform, morphologically distinct VT occurring in 52 patients. Of the 80 tachycardias receiving single VE, 41 were reset and 8 terminated. The corrected coupling interval of single VE first causing resetting was 0.81 +/- 0.08 compared with 0.66 +/- 0.06 for termination (p less than 0.001). Forty-two tachycardias received double VE with 33 being reset and 13 terminating. The corrected coupling interval of double VE at which resetting was first seen was 0.86 +/- 0.08 compared with 0.73 +/- 0.05 for termination (p less than 0.001). If the longest corrected coupling interval causing resetting was greater than or equal to 0.75, then 7 of 34 tachycardias terminated with single VE and 13 of 31 terminated with double VE compared with only 1 of 46 terminating with single VE and 0 of 10 with double VE if resetting was not observed by a corrected coupling interval of 0.75 (p less than 0.01 and p less than 0.02, respectively). If the longest corrected coupling interval at which resetting occurred was greater than or equal to 0.75, the predictive value for VT termination was 21% with single VE and 42% with double VE compared with only 2% with single VE and none with double VE if resetting was not observed by this corrected coupling interval.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiac Pacing, Artificial↗

PubMed1978

Isolation of protein subunits by coupling to an insoluble matrix: analysis of interactions and application to G-actin.

A criterion has been devised for the assessment of intermolecular contacts between protein subunits coupled to a gel matrix. After reversible coupling by way of disulfide groups to Sepharose 4B, the system is exposed to a bifunctional reagent. The protein is then released by reduction, and the proportion of dimers and higher oligomers formed by cross-linking is determined by gel electrophoresis, followed by densitometry of the stained gels. Experiments were performed with G-actin coupled to dithio-2-dipyridyl Sepharose 4B. At low concentration of coupled protein, no species other than the monomer were obtained; under these conditions any intermolecularly cross-linked species would represent pre-existing associated states coupled to the matrix. At higher protein concentrations dimers and higher species progressively appear. Their proportion is much higher than predicted on the basis of a random statistical distribution of coupled molecules throughout the accessible internal volume of the matrix. It follows that protein-protein contacts can occur either because of high flexibility of the polysaccharide chains of the matrix, or because the protein is partly (but not wholly) present as a shell on the surface of the beads. With Affi-gel 10, which has N-hydroxysuccinimide ester coupling groups, similar experiments were performed, taking advantage of the degradation of the matrix under relatively mild acid conditions. In this case the degree of cross-linking was much lower than in the Sepharose 4B system at the same protein concentration. However, this medium proved unsatisfactory for the measurement of interactions of the bound actin with other muscle proteins present in the mobile phase. The results with Sepharose 4B support the validity of previous studies on interaction of monomeric actin with other muscle proteins.

Actins↗

PubMed1978

Evidence for a catalytic function of the coupling factor 1 protein reconstituted with chloroplast thylakoid membranes.

The effects of tentoxin on the ATPase activities of coupling factor 1 proteins (CF1) and photophosphorylation with isolated chloroplasts and chloroplasts reconstituted with coupling factor proteins have been examined. 1. The calcium-dependent ATPase activities of coupling factors isolated from spinach, lettuce and Nicotiana otophora are completely inhibited by tentoxin. The ATPase activities of coupling factors isolated from Nicotiana tabacum and Nicotiana knightiana are not affected by tentoxin. 2. Phenazine methosulfate-catalyzed cyclic photophosphorylation with chloroplasts isolated from spinach, lettuce and N. otophora is completely inhibited by tentoxin, whereas chloroplasts isolated from N. knightiana and N. tabacum are relatively insensitive to tentoxin. 3. Spinach chloroplasts, partially depleted in CF1, can be reconstituted with coupling factors isolated from a wide variety of plants including lettuce, radish, N. tabacum, N. knightiana and N. otophora. 4. Spinach chloroplasts reconstituted with spinach, lettuce and N. otophora CF1 retain their sensitivity to tentoxin; however, when reconstituted with N. knightiana and N. tabacum coupling factor proteins, a significant fraction of the reconstituted rate remains tentoxin insensitive. These data are interpreted as evidence that coupling factors that reconstitute with spinach thylakoid membranes have both a catalytic and structural function.

Adenosine Triphosphatases↗

PubMed1980

Electrostatic control of chloroplast coupling factor binding to thylakoid membranes as indicated by cation effects of electron transport and reconstitution of photophosphorylation.

1. Increase in electron transport rate and the decay rate of the 518 nm absorption change, induced by EDTA treatment, is prevented by cations. The order of effectiveness is C3+ > C2+ > C+. 2. In this respect methyl viologen is an effective divalent cation in addition to its action as an electron acceptor. 3. Complete cation irreversible EDTA-induced uncoupling occurs in the dark in 2 min. Light greatly stimulates the rate of uncoupling by EDTA. It is concluded that the uncoupling is due to release of coupling factor I from the thylakoid membrane. 4. Binding of purified coupling factor I to coupling factor I-depleted thylakoids can be achieved with any cation. The order of effectiveness is C3+ > C2+ > C+, reconstituted thylakoids are active in photophosphorylation regardless of the cation used for coupling factor I binding. 5. The marked difference in the concentration requirements for cation effects on 9-aminoacridine fluorescence yield and for prevention of uncoupling by EDTA indicate that coupling factor I and its binding site have a lower surface charge density than the net surface charge density of the thylakoid membrane. 6. It is concluded that coupling factor I binding only occurs when negative charges on coupling factor I and its binding site are electrostatically screened by cations. 7. Previously reported examples of uncoupling by low ionic conditions are discussed in relation to the basic concepts of diffuse electrical layer theory.

Cations↗

PubMed1980

Sulfhydryl groups in photosynthetic energy conservation. VI. Subunit distribution of sulfhydryl groups and disulfide bonds in chloroplast coupling factor and ATPase activity.

The subunit distribution of sulfhydryl groups and disulfide bonds of spinach chloroplasts coupling factor I has been determined. Native coupling factor I with a latent ATPase activity has eight sulfhydryl groups distributed 4 : 2 : 0 : 0 : 2 in the alpha, beta, gamma, delta and epsilon subunits, respectively. Heat treatment of coupling factor I, in addition to the activation of its ATPase activity, induces a dithiol-disulfide interchange between the gamma and the alpha subunit, changing the sulfhydryl groups' distribution to 2 : 2 : 2 : 0 : 2. Reduction of disulfide bonds of coupling factor I by dithioerythritol during heat treatment gives a subunit distribution of 4 : 4 : 4 : 0 : 2, suggesting that native coupling factor I has three disulfide bonds, two in the gamma subunit and one in one of the beta subunits. The results suggest an asymmetric redox state of some of the subunits of coupling factor I and an asymmetric positioning of some of them in the molecular structure of coupling factor I.

Adenosine Triphosphatases↗

PubMed1990

Comparative studies of the preparation of immunoliposomes with the use of two bifunctional coupling agents and investigation of in vitro immunoliposome-target cell binding by cytofluorometry and electron microscopy.

The two coupling agents SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate) and SATA (N-succinimidyl-S-acetylthioacetate) were compared in their efficiency and feasibility to couple monoclonal antibodies (Abs) via thioether linkage to liposomes functionalized by various lipophilic maleimide compounds like N-(3-maleimidopropionyl)-N2-palmitoyl-L-lysine methyl ester (MP-PL), N-(3-maleimidopropionyl)phosphatidylethanolamide (MP-PE), N6-(6-maleimidocaproyl)-N2-palmitoyl-L-lysine methyl ester (EMC-PL), and N-(6-maleimidocaproyl)phosphatidylethanolamine (EMC-PE). The composition of the liposomes was soy phosphatidylcholine (SPC), cholesterol, maleimide compounds and alpha-tocopherol (1:0.2:0.02:0.01, mol parts), plus N4-oleylcytosine arabinoside (NOAC) as cytostatic prodrug (0.2 mol parts) and a new, lipophilic and highly fluorescent dye N,N'-bis(1-hexylhfetyl)-3,4:9,10-perylenebis(dicarboximid ) (BHPD, 0.006 mol parts). From the maleimide derivatives MP-PL was the most effective in terms of preservation of the coupling activity in dependence of liposome storage. The coupling of the monoclonal A B8-24.3 (mouse IgG2b, MHC class I, anti H-2kb) and IB16-6 (rat IgG2a, anti B16 mouse melanoma) to the drug carrying liposomes was more effective and easier to accomplish with SATA as compared to SPDP. Coupling rates of 60-65% were obtained with SATA at molar ratios of 12 SATA:1 Ab:40 maleimide spacer groups on the surface of one liposome. The highest coupling rates with SPDP were obtained at the ratio of 24 SPDP:1 Ab:40 liposomal maleimide groups, with an Ab binding efficiency of only 20-25%. The optimal in vitro binding conditions to specific target cells (EL4 for B8-24.3-liposomes and B16-F10 for IB16-6-liposomes) were determined by cytofluorometric measurement of the liposomal BHPD fluorescence with SATA linked Abs. Optimal immunoliposome binding to specific epitopes on the target cells was achieved with 1-2 Ab molecules coupled to one liposome, with immunoliposome concentrations of 20-130 nM and with a small incubation volume of 0.3-0.4 ml. The specificity of the binding of B8-24.3-liposomes to EL4 target cells was visualized by scanning electron microscopy. Antibody mediated endocytic uptake of immunoliposomes could be demonstrated by transmission electron microscopy.

Animals↗

PubMed1995

A novel strategy affords high-yield coupling of antibody Fab' fragments to liposomes.

A new assay for the production of reactive sulfhydryl-bearing antibody Fab' fragments has been utilized to develop conditions affording high efficiencies of coupling of mouse and rabbit IgG-derived Fab' fragments to lipid vesicles containing maleimidyl-functionalized phospholipids. Cysteine and mercaptoethylamine, but not dithiothreitol, reduce antibody F(ab')2 to Fab' fragments in very good yields under conditions where overreduction to heavy and light chains is minimized. Surprisingly, however, a large fraction of the Fab' fragments generated under these conditions can lack maleimide-reactive sulfhydryl groups, as demonstrated using a maleimidyl-poly(ethylene glycol) conjugate to shift selectively the electrophoretic mobility of the reactive sulfhydryl-bearing Fab' fragments. After modification of F(ab')2 reduction conditions specifically to maximize the yield of the latter fraction, it is possible to achieve high and very reproducible coupling of functional Fab' fragments to liposomes (equivalent to coupling of ca. 70% of total input protein and almost 100% of the reactive sulfhydryl-bearing Fab' fraction). A novel phospholipid-poly(ethylene glycol)-maleimide 'anchor' allows particularly efficient coupling of Fab' fragments to liposomes, even using relatively low liposome concentrations and molar percentages of the liposome-incorporated 'anchor' species. These results demonstrate that with appropriate optimization of the conditions for Fab' production and liposome coupling, Fab' fragments can be coupled to liposomes with efficiencies comparable to or exceeding those reported for coupling of intact antibodies. These results should facilitate the wider use of Fab' fragments as a potentially advantageous alternative to intact antibodies for liposomal targeting in various applications.

Animals↗

PubMed1986

The pattern of functional coupling of brain regions in the awake rat.

The functional interactions among many regions of the rat brain were characterized simultaneously by using a new method for analyzing regional cerebral metabolic rates for glucose (rCMRglc). Regional CMRglc, a measure of regional functional activity, was determined in 68 brain regions of 24 awake, male, 3-month-old Fischer rats by the quantitative autoradiographic [14C]deoxyglucose technique. Because rCMRglc for each region varies among subjects, the functional coupling of two regions can be assessed by determining whether their metabolic rate change in a coordinated manner from rat to rat. Positive coupling arises if, for each rat, higher rCMRglc in one region is accompanied by a higher metabolic rate in another area; likewise, regions whose metabolic rates vary inversely are negatively coupled. A pairwise coupling can be quantified by calculating the linear correlation coefficient for points whose coordinates represent rCMRglc values for the two regions in each of the 24 rats. Correlation coefficients were calculated for each possible region pair among the 68 areas studied. The technique of partial correlation was used to weight equally the contribution of each rat to the correlation coefficient, regardless of its overall mean CMRglc. In general, highly positive couplings (R greater than 0.5) were found between adjacent or nearby regions, whereas distant region pairs often were coupled negatively (R less than -0.5). A large number of positive couplings were found between left-right homologous regions and between neocortical areas. Similar findings in humans, obtained by positron emission tomography, have been reported. This technique uses inter-animal variation in rCMRglc to demonstrate functional interactions among brain areas, thus providing a method to explore anatomical-functional linkages in a given experimental state.

Animals↗