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Macrophages from C57BL/6J mice, an animal susceptible to atherosclerosis, were chosen as target cells to study changes in the transmembrane Ca2+ gradient during the formation of macrophage-derived foam cells. The transmembrane Ca2+ gradients of single living cells were examined-using Fura-2/AM combined with Fluo-3/AM by laser scanning confocal microscopy. Exposure to Oxidized Low Density Lipoprotein, decreases the Ca2+ gradient across macrophage plasma membrane, but increases that across the nuclear membrane. The altered transmembrane Ca2+ gradients could induce the up-regulation of scavenger receptor in macrophages, resulting in the formation of foam cells.

The relationship between Ca2+ transport and energy transduction of myocardial mitochondria in the presence of reactive oxygen species was investigated. Following treatment with oxygen free radicals [superoxide(O2•) or hydroxyl radical (•)OH], lipid free radicals in myocardial mitochondrial membrane could be detected by using the method of EPR spin trap. Simultaneously there were obvious alterations in the free Ca2+ ([Ca2+]m) in the mitochondrial matrix; the physical state of membrane lipid; the efficiency of oxidative phosphorylation (ADP/O); the value of the respiratory control ratio (RCR); and the membrane potential of the inner membrane of myocardial mitochondria. If the concentrations of reactive oxygen species were reduced by about 30%, the alterations in the physical state of the membrane lipid and energy transduction of myocardial mitochondria were not observed, but the changes in Ca2+ homeostasis remained. We conclude that Ca2+ transport by myocardial mitochondria is more sensitive to agents such as (O2•) or •OH, etc. than are oxidation phosphorylation and the respiratory chain.

It was shown that apocytochrome c was less insertion into monolayers from outer mitochondrial membrane lipids than into those from microsomal membrane lipids; the alpha-helix content of apocytochrome c induced by small unilamellar vesicles prepared from outer mitochondrial membrane lipids was less than by those from microsomal membrane lipids; the import efficiency of apocytochrome c into large unilamellar vesicles from outer mitochondrial membrane lipids was also lower than into those from microsome membrane lipids. No specific affinity between apocytochrome c and outer mitochondrial membrane lipids could be found. Import of apocytochrome c across the intact mitochondria, sealed outer mitochondrial membrane and microsome membrane vesicles was compared. Results showed that apocytochrome c was accumulated only in mitochondria, but not the other two kinds of vesicles.

This review will focus on the recent advance in the study of effect of transmembrane Ca2+ gradient on the function of membrane proteins. It consits of two parts: 1. Transmembrane Ca2+ gradient and sarcoplasmic reticulum Ca2+-ATPase; 2. Effect of transmembrane Ca2+ gradient on the components and coupling of cAMP signal transduction pathway. The results obtained indicate that a proper transmembrane Ca2+ gradient may play an important role in modulating the conformation and activity of SR Ca2+-ATPase and the function of membrane proteins involved in the cAMP signal transduction by mediating the physical state change of the membrane phospholipids.

The conformational states of Ca2+-ATPase in sarcoplasmic reticulum (SR) vesicles with or without a thousand-fold transmembrane Ca2+ gradient have been studied by fluorescence spectroscopy and fluorescence quenching. In consequence of the establishment of the transmembrane Ca2+ gradient, the steady-state fluorescence results revealed a reproducible 8% decrease in the intrinsic fluorescence while time-resolved fluorescence measurements showed that 13 tryptophan residues in SR · Ca2+-ATPase could be divided into three groups. The fluorescence lifetime of one of these groups increased from 5.5 ns to 5.95 ns in the presence of a Ca2+ gradient. Using KI and hypocrellin B (a photosensitive pigment obtained from a parasitic fungus, growing in Yunnan, China), the fluorescence quenching further indicated that the dynamic change of this tryptophan group, located at the protein-lipid interface, is a characteristic of transmembrane Ca2+ gradient-mediated conformational changes in SR · Ca2+-ATPase.

Divalent cations play an important role in the functions of biomembranes. This review deals with three topics: (1) Mg2+-mediated change in physical state of phospholipid induces conformation and activity change of reconstituted mitochondrial H+-ATPase, (2) a proper transmembrane Ca2+ gradient is essential for the higher enzymatic activity of adenylate cyclase, and (3) role of transmembrane Ca2+ gradient in the modulation of reconstituted sarcoplasmic reticulm Ca2+-ATPase activity.

Stimulation of the host immune system is crucial in cancer treatment. In particular, nonspecific immunotherapies, when combined with other traditional therapies such as radiation and chemotherapy, may induce immunity against primary and metastatic tumors. In this study, we demonstrate that a novel, non-toxic immunoadjuvant, glycated chitosan (GC), decreases the motility and invasion of mammalian breast cancer cells in vitro and in vivo. Lung metastatic ratios were reduced in 4T1 tumor-bearing mice when intratumoral GC injection was combined with local high-intensity focused ultrasound (HIFU) treatment. We postulate that this treatment modality stimulates the host immune system to combat cancer cells, as macrophage accumulation in tumor lesions was detected after GC-HIFU treatment. In addition, plasma collected from GC-HIFU-treated tumor-bearing mice exhibited tumor-specific cytotoxicity. We also investigated the effect of GC on epithelial-mesenchymal transition-related markers. Our results showed that GC decreased the expression of Twist-1 and Slug, proto-oncogenes commonly implicated in metastasis. Epithelial-cadherin, which is regulated by these genes, was also upregulated. Taken together, our current data suggest that GC alone can reduce cancer cell motility and invasion, whereas GC-HIFU treatment can induce immune responses to suppress tumor metastasis in vivo.

Chicken apocytochrome c has been shown to possess a much stronger tendency to fold spontaneously in aqueous solution than the equivalent enzyme from other species. In the present work, the amino acid that determines its folding ability was elucidated by site-directed mutagenesis. Wild-type chicken apocytochrome c and three mutants V92A, S103A, and V92A/S103A were expressed in Escherichia coli. The wild-type apoprotein and S103A exhibited the same folding property during dialysis renaturation processes as that chemically prepared from chicken cytochrome c, while those containing V92A mutation did not. Quantitative studies by 2,2,2-trifluoroethanol (TFE) and sodium perchlorate (NaClO4) titration demonstrated that the V92A mutation decreased the helix content that could be induced and confirmed that valine 92 is the major determinant of the folding propensity of chicken apocytochrome c. Furthermore, CD spectra, turbidity measurements, and a translocation assay on a model membrane system showed that the V92A mutation also drastically altered the conformation of apocytochrome c after being incorporated into lipid bilayer and decreased the aggregation of phospholipid vesicles after association of the apoprotein, thus rendering the molecule more competent for translocation across the membrane. Our results showed that a single amino acid substitution could radically alter the folding propensity of an unfolded polypeptide chain and thus influence the conformation following its insertion into phospholipid bilayer.

Chicken apocytochrome c gene with correct reading frame was easily cloned through excision by polymerase chain reaction of the intron in the genomic clone of chicken cytochrome c gene, and was successfully overexpressed in Escherichia coli by cloning into expression vector pET-3d under the control of T7 promoter. Expressed protein can amount to as high as 40% of the total protein and mainly presents as inclusion body. Purification of chicken apocytochrome c from the inclusion body and characterization by SDS-PAGE, isoelectric focusing electrophoresis, and amino acid analysis showed that the purified apocytochrome c is identical to that prepared from chicken heart cytochrome c by chemically depletion of heme.

In contrast to horse heart apocytochrome c, the chicken one showed quite different folding propensity as titrated by NaCl at different pH. At pH 2.0, folding behaviour of both apocytochrome c are essentially similar; while at pH higher than 4.0, chicken heart apocytochrome c has much enhanced propensity to fold and aggregate, as was shown by circular dichroism spectra, intrinsic fluorescence and non-denatured polyacrylamide gel electrophoresis. Hydrophobic chromatography demonstrated much higher hydrophobicity of chicken heart apocytochrome c, thus strongly suggested that it is the hydrophobic interaction that stabilize the 'Molten Globule' like, partially-folded structure of chicken heart apocytochrome c at neutral pH.

In contrast to the horse heart apocytochrome c, the chicken heart apocytochrome c underwent a conformational change from random coil to partial folding during a renaturation process. When the apocytochrome horse heart and that of chicken heart c were subjected to a translocation assay in vitro using large trypsin-enclosed unilamellar vesicles from soybean phospholipids, the ability of the chicken heart apocytochrome c to penetrate into the liposomes was found to decrease markedly with the renaturation procedure, while that of horse heart apocytochrome c remained relatively constant. Observations from circular dichroism measurement on the induction of secondary folding of these two species of apocytochrome c upon interaction with soybean phospholipid vesicles suggested that a more flexible structure of apocytochrome c embedded in the lipid matrix be required for its efficient translocation across the bilayer.

Sarcoplasmic reticulum (SR) vesicles with (1000 folds) or without transmembrane Ca2+ gradient have been prepared. Different fluorescence probes (DPH, TMA-DPH and n-AS), were used to determine the effect of transmembrane Ca2+ gradient on the lipid fluidity both in outer and inner layer of Ca(2+)-ATPase-containing SR vesicles. The results showed that transmembrane Ca2+ gradient could significantly decrease the fluidity of the inner layer of SR membrane, while no obvious change was monitored in the outer layer. This may be deduced that Ca(2+)-ATPase might be modulated mainly by the transmembrane Ca2+ gradient-mediated alteration of physical state of phospholipid in the inner layer of SR membrane.

We have previously reported that transmembrane Ca2+ gradient-mediated changes in lipid fluidity could modulate the conformation and enzyme activity of sarcoplasmic reticulum (SR) Ca(2+)-ATPase. The aim of this paper is to explore the specificity of transmembrane Ca2+ gradient-mediated modulation of SR Ca(2+)-ATPase. The results showed that such specificity exhibited in two aspects: 1. The modulation could not be ascribed to transmembrane potential resulted from the transmembrane Ca2+ gradient, Dissipation of transmembrane potential by FCCP (carbonylcyanide-p-trifluoromethoxyphenylhydrazone) could not affect the activity of SR Ca(2+)-ATPase. 2. Transmembrane Sr2+ gradient had little effect on the enzyme activity of SR Ca(2+)-ATPase. A significant difference between the effect of transmembrane Ca2+ and Sr2+ gradient on the lipid fluidity was detected in the middle region of bilayer of Ca(2+)-ATPase incorporated proteoliposomes using a set of n-AS [n-(9-anthroyloxy) fatty acids] fluorescence polarization probes. It is known that Ca2+ binding domain of SR Ca(2+)-ATPase is just located in the middle region of bilayer, hence it may be deduced that possibly, membrane lipids are involved in transmembrane Ca2+ gradient-mediated modulation of Ca(2+)-ATPase.

Chemically-prepared chicken heart apocytochrome c with different unfolded states could be obtained during the renaturation process. They exhibited distinct circular dichroism patterns designated as Apo C1 (random coiled), Apo C2 (less ordered) and Apo C3 (more ordered). This characteristic is unique to chicken heart apocytochrome c while compared with its counterparts from Candida krusei, tuna heart or horse heart and promises the emergence of much more detail of correlation of translocation with unfolded states of apocytochrome c. When chicken heart apocytochrome c was subjected to a translocation assay in vitro using trypsin-enclosed large unilamellar vesicles from soybean phospholipids, the ability of the protein to penetrate into the liposomes was found to follow the order of Apo C1Apo C2Apo C3. Conformational alterations of Apo C1, Apo C2 and Apo C3 in association with soybean phospholipid vesicles shown by circular dichroism measurement demonstrated that Apo C1 bound to phospholipids existed in a more loosely folded conformation than Apo C2 and Apo C3. We propose that a more flexible structure of apocytochrome c following the interaction with phospholipids is required for its efficient translocation across the bilayer.

The translocation ability of two different species of apocytochrome c(horse heart, Candida krusei) across the soybean phospholipid vesicles decreased in the order: C. kruseihorse heart. Theoretical calculations of the amphiphilicity of their N- and C-terminal alpha-helix formation and the release experiment using calcein-enclosed soybean phospholipid vesicles showed that there was no direct relevance between their import ratio and the amphiphilic helicity. On the other hand, taking the advantage of circular dichroism technique, a more loosely folded structure of C. krusei following the interaction with soybean phospholipid vesicles was observed which should be responsible for the difference in translocation rate.

Adenylate cyclase from bovine brain cortex was reconstituted into asolectin liposomes with (500-fold) or without transmembrane Ca2+ gradient. The enzyme activity of four types of proteoliposomes (the active center of enzyme exposing outside) was compared. The highest adenylate cyclase activity was observed in the vesicles with outside lower Ca2+ concentration (approximately 10(-6) mol/L, similar to the physiological condition). If the transmembrane Ca2+ gradient was in the inverse direction (i.e. outside higher Ca2+ concentration, 0.5 mmol/L), a lowest enzymatic activity would appear. The difference in enzymatic activity between the two types of proteoliposomes could be diminished following the addition of Ca2+ ionophore A23187. Proteoliposomes without transmembrane Ca2+ gradient exhibited intermediate activities. The conformation difference of adenylate cyclases in the above-mentioned proteoliposomes was also detected by measuring intrinsic fluorescence and fluorescence quenching with KI.

Hybridomas producing monoclonal antibodies (McAb) directed against Trichomonas vaginalis have been produced by fusing NSI myeloma cells with spleen cells of BALB/c mice immunized with Trichomonas vaginalis. IFA technique was used to test the binding activity of four McAbs produced. The McAb belonged to the IgG subtypes IgG1 (2A2, 2A4 McAb), IgG3 (2H9 McAb) and IgG 2b (2A12 McAb). Three McAbs, designated 2A2, 2A4, 2A12, reacted with a surface membrane component of live Trichomonas vaginalis. One (2A12) of them produced complement-dependent cytolysis of the parasites. Others (2A2,2A4) produced complement-independent cytotoxicity of the parasites. 2H9 McAb which reacted with the nucleus of the organisms did not agglutinate the parasites. The four McAbs which did not have cross reaction with some protozoa of Zoomastigophorea species were specific antibodies against Trichomonas vaginalis. (Figs. 1-3).

During the process of reconstitution of pig heart mitochondrial H+-ATPase on liposomes by cholate dialysis method, 1 mM Mg2+ in the dialysis medium can obviously increase activities of the reconstituted H+-ATPase (32Pi-ATP exchange, enzyme activity and its sensitivity to oligomycin or DCCD and ATP-dependent ANS 1) fluorescence). Besides Mg2+, effects of other divalent cations and spermidine on the H+-ATPase reconstitution have been compared. The effectiveness of the divalent cation activation of 32Pi-ATP exchange of the reconstituted H+-ATPase, decreased in the order: Mg2+ greater than Ca2+ greater than Mn2+ greater than Sr2+, while that of increasing the sensitivity to the inhibitors: Ca2+ greater than Mg2+ greater than Mn2+ greater than Sr2+. No significant effect on the H+-ATPase reconstitution has been found with Cd2+, Zn2+ and spermidine3+. Mechanism of action of the divalent cations on reconstitution has been discussed.

During reconstitution of pig heart mitochondrial H(+)-ATPase in soybean phospholipid liposomes by the cholate dialysis method, Mg2+ greatly enhanced 32Pi-ATP exchange activity, ATPase activity and the sensitivity to oligomycin or DCCD of the reconstituted enzyme complex. The effect of Mg2+ on the lipid packing or fluidity of the reconstituted proteoliposomes was measured by means of spin labels, fluorescent probes and pyrene excimer formation efficiency. A difference in fluidity seemed to be localized near the polar faces of lipid bilayers of the reconstituted enzyme complex. Fluidity was less in the presence of Mg2(+)-containing and the Mg2(+)-'free' samples. Based on the results obtained a hypothetical scheme was proposed for Mg2(+)-mediated change in the physical state of phospholipid modulates incorporating H(+)-ATPase in liposomes. It postulated that Mg2+ may play a role in altering the lipid fluidity of the bilayers, which would induce a change of conformation of F0 portion (buried in the lipid core) of H(+)-ATPase complex. Such change could be transmitted to the soluble F1 portion, the conformation of which is in turn altered, resulting in higher enzymatic activity. Such an assumption was further supported by the results of a series of biochemical and biophysical experiments. Similar to its effect in the reconstitution of porcine heart mitochondrial H(+)-ATPase in liposomes, Mg2+ may also enhance the enzyme activity of reconstituted cytochrome C oxidase, porcine kidney medulla Na,K-ATPase, Ca-ATPase from rabbit sarcoplasmic reticulum and chloroplast H(+)-ATPase, in liposomes. It may be inferred that the structure and function of many membrane proteins are similarly modulated by Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS)

A partially purified fraction SVc and a purified homogeneous polypeptide SVIII were isolated from the scorpion (Buthus martensii Kashi) venom, collected in Shan Dong Province of China. SVc decreased the RCR, ADP/O and Qo2 of the rat brain mitochondria. It also decreased the cytochrome oxidase activity and increased the membrane lipid fluidity of the mitochondria. Effect of scorpion venom on the rat heart mitochondria was somewhat different from that of rat brain mitochondria. SVc also decreased RCR, ADP/O and increased the membrane lipid fluidity of heart mitochondria. However, the Qo2 and cytochrome oxidase activity were increased. SVIII has a similar effect on the rat brain and heart mitochondria, but its concentration used is only 1/10 of the effective concentration of SVc.