Expression and purification of metabotropic glutamate receptor 7 peptides.

Metabotropic glutamate receptors (mGluRs) influence a variety of second-messenger systems and ion channels. The C-terminal region of group III mGluRs interacts with the Ca(2+)-binding protein calmodulin (CaM). We intend to study the interaction between Ca(2+)/CaM and the CaM-binding motifs within mGluR(7), which is a group III mGluR. We established a recombinant protein expression and purification system for nuclear magnetic resonance (NMR) analysis of mGluR(7) peptides using Escherichia coli. Peptides of mGluR(7) conjugated to an affinity tag sequence were constructed, and protocols for expression and purification were optimized. To suppress non-specific enzymatic cleavage, the mGluR(7) fusion peptide was bound to Ca(2+)/CaM before enterokinase cleavage. This complex method for precise enzymatic reactions may be applicable for the recombinant preparation of a wide variety of peptides.

Stability of asymmetric phospholipid membranes.

Bilayers (black films) composed of phosphatidylserine are unstable under conditions of asymmetric distribution of calcium or hydrogen ions with respect to the membrane. Addition of calcium ions to the solution (100 millimolar sodium chloride, pH 7.0) on one side only, produces lowering of the direct-current resistance and results in breaking of the membrane. However, with calcium ions on both sides the membranes are stable and show very high electrical resistance.

Glycation and phosphorylation of alpha-lactalbumin by dry heating: effect on protein structure and physiological functions.

Alpha-lactalbumin (alpha-LA) was glycated with maltopentaose (MP) through the Maillard reaction (MP-alpha-LA) and subsequently phosphorylated by dry heating in the presence of pyrophosphate to investigate its structure and physiological functions. Glycation occurred effectively, and the sugar content of alpha-LA increased by approximately 22.3% through the Maillard reaction. The phosphorylation of MP-alpha-LA was enhanced with an increase in the dry-heating time from 1 to 5 d, and the phosphorous content of MP-alpha-LA increased by approximately 1.01% by dry heating at pH 4.0 and 85 degrees C for 5 d in the presence of pyrophosphate. The electrophoretic mobility of alpha-LA increased with an increase in the phosphorylation level. The circular dichroism spectra showed that the change in the secondary structure of the alpha-LA molecule by glycation and subsequent phosphorylation was slight. However, the Trp fluorescence intensity was increased by phosphorylation after glycation. In addition, the differential scanning calorimetry thermograms of alpha-LA showed that the denaturation temperature of MP-alpha-LA was decreased by phosphorylation. These results indicated that molten (partially unfolded) conformations of alpha-LA were formed by dry heating in the presence of pyrophosphate after glycation. The anti-alpha-LA antibody response was significantly reduced by glycation and subsequent phosphorylation. The suppressive effect of alpha-LA on the production of proinflammatory cytokines such as IL-6 and tumor necrosis factor-alpha from THP-1 cells after stimulation with lipopolysaccharide was significantly enhanced by glycation with MP and was further enhanced by phosphorylation after glycation. The Ca phosphate-solubilizing ability of alpha-LA was enhanced by phosphorylation. The apoptotic activity of alpha-LA was reduced by glycation and subsequent phosphorylation. These results suggest that phosphorylation by dry heating in the presence of pyrophosphate after glycation with MP through the Maillard reaction is a useful method for improvement of the physiological functions of alpha-LA.

[Repair of paravalvular leak after a third mitral valve replacement].

This study reports on a 57-year-old woman who underwent a 3rd mitral valve replacement and presented with complaints of fatigue. Laboratory examination revealed severe hemolytic anemia, and trans-esophageal echocardiography revealed a paravalvular leak (PVL) around the prosthetic valve at the posterior trigone in the mitral position. PVL was regarded as the cause of hemolytic anemia. At surgery, a small tissue defect was detected around the calcified posterior trigone of the mitral annulus with no evidence of infective endocarditis. The mitral PVL was successfully repaired with suture closure of the annular defect. The postoperative course was uneventful: postoperative echocardiography revealed no evidence of PVL, and the hemolytic anemia subsided.

Colonic J-pouch decreases bowel frequency by improving the evacuation ratio.

BACKGROUND/AIMS: To compare the functional outcome of ultra-low anterior resection for rectal cancer with colonic J-pouch reconstruction with that of straight reconstruction. METHODOLOGY: Twenty-three patients who underwent ultra-low anterior resection with or without J-pouch reconstruction underwent bowel transit study, videodefecography, and answered a questionnaire survey 4 months and 1 year after surgery. Eleven healthy subjects underwent similar testing as controls. RESULTS: Patients with a J-pouch had less frequent stools than patients with straight reconstruction 4 months after surgery (p<0.05), but the two groups were similar at 1 year. Bowel transit time was similar at both study points. The evacuation ratio was higher after J-pouch than straight reconstruction 4 months after surgery (p<0.05). However, the ratio improved in the straight group, and no difference existed at 1 year. Colonic contraction was seen only near the anastomosis 4 months after surgery, but the contraction proximal to the anastomosis improved over the next 8 months. CONCLUSIONS: J-pouch reconstruction facilitates evacuation by improving the evacuation ratio. Although straight anastomosis caused excessive stool frequency 4 months after surgery, colonic function continued to improve and was comparable with J-pouch and straight reconstruction 1 year after surgery because the contraction ratio proximal to the anastomosis improved.

[Surgical fixation of the ribs for flail chest injuries].

The record of 20 patients presenting with flail chest injury from 1998 to 2005 was reviewed to determine surgical indication and timing. There were 4 groups with each indication as followed: 1) 8 patients with surgical indication for injury regions other than fractured ribs, 2) 5 without improvement of flail chest after internal pneumatic stabilization for more than 10 days, 3) 4 performed surgical fixation positively for flail chest with respiratory failure, 4) 3 with strong deformation of the thorax without respiratory failure. Eight patients (40%) required artificial respiration for more than 6 days after surgical stabilization. The reasons of prolonged artificial respiration included unconsciousness in 4 patients, pneumonia in 2, and others in 2. In the group consisting of 8 patients taking more than 6 days to be extubated after surgical fixation, the injury severity score (ISS) was significantly higher (p = 0.006) than that of the other group. In patients with no improvement of flail chest after internal pneumatic stabilization for more than 10 days, surgical fixation reduces the period of internal pneumatic stabilization and the risk of pneumonia. For the elderly who can develop complications easily, early indication of surgical fixation should be considered. In patients with unconsciousness or ISS > or = 25, the extubation delays frequently after surgical fixations.

[Ascending aortic aneurysm following aortic valve replacement due to aortitis syndrome; report of a case].

A 60-year-old woman had previously undergone aortic valve replacement for aortic regurgitation. As the aortic wall was elastic hard, inflammatory change was suspected; therefore, we undertook a partial biopsy of the ascending aortic wall and the intraoperative pathological specimens were compatible with aortitis syndrome. As there was no active inflammatory change, she was diagnosed as inactive aortitis syndrome and steroid therapy was not applied. Seven years later, a follow-up computed tomography (CT) showed an ascending aortic aneurysm of 65 mm in diameter. Aortic root replacement was planned based on a clinical diagnosis of an aneurysm of the ascending aorta. The patient was discharged without complication 21 days after surgery. It is possible that an inactive stage of aortitis may lead to late dilatation of the ascending aorta; therefore, careful postoperative follow-up is necessary in such cases.

Adsorption of local anesthetics on phospholipid membranes.

In order to elucidate various types of adsorption modes of local anesthetics in membranes, a study of local anesthetic adsorption on lipid membranes was made by measuring electrophoretic mobility of phospholipid vesicles in the presence of local anesthetics of various concentrations in the vesicle suspension solution. The amounts of local anesthetics to be adsorbed on the membrane surface were deduced from the electrophoretic mobility of a phosphatidylcholine vesicle at various concentrations of the cationic form of local anesthetics. The order of surface adsorption of local anesthetic was dibucaine greater than tetracaine greater than procaine. A surface partition coefficient, Ks = 1/ACs, was introduced, where A is the membrane surface area per local anesthetic molecule adsorbed and Cs the surface concentration of local anesthetics. The amounts of local anesthetic adsorbed on phosphatidylserine membrane were much greater than that of the phosphatidylcholine membrane. It was deduced that the major factor for this large adsorption was due to the enhancement of cationic forms of local anesthetic concentrations at the charged membrane surface. Divalent cations inhibited such surface adsorption of local anesthetics by reducing surface concentrations of local anesthetics where the surface potential of the negatively charged membrane surface was influenced by the presence of divalent cations in the solution as well as by the reduction of fixed surface charges due to divalent cation binding. Some association modes of local anesthetics on nerve membranes are discussed with the results obtained in the above adsorption study.

A mechanism of divalent ion-induced phosphatidylserine membrane fusion.

A mechanism for the divalent cation-induced membrane fusion of phosphatidylserine membranes is proposed. Fusion was followed by the Tb/DPA (dipicolinic acid) assay, monitoring the fluorescent intensity for mixing of the internal aqueous contents of unilamellar lipid vesicles, and the threshold concentrations required for various divalent cations to induce membrane fusion were determined from the fluorescence spectrum of the lipid vesicle suspension with respect to various concentrations of divalent ions. Also, the surface tension of monolayers made of the same lipids as used in the fusion experiments was measured with respect to the variation of divalent cation concentrations. The surface tension increase in the monolayer, induced by changing divalent ion concentrations from zero to a concentration which corresponded to its threshold concentration to induce vesicle membrane fusion, was the same (approx. 8 dyn/cm) for all divalent ions used. From these experimental data and theory concerning ion binding to the membrane, it is deduced that the main cause of divalent cation-induced membrane fusion of phosphatidylserine membranes is the degree of increased hydrophobicity (surface tension increase) of the membrane surface, which results from the binding of cations to acidic phospholipid membrane surfaces. Some discussion on the molecular mechanism of phospholipid membrane fusion is given.

Effects of cations and polyamines on the aggregation and fusion of phosphatidylserine membranes.

Effects of various metal cations and polyamines on aggregation and fusion of phosphatidylserine vesicles and their associated physicochemical properties (such as surface tension and vesicle electrophoretic mobility) have been studied. It was found that metal polycations and hydrogen ion caused an increase in the surface tension of a phosphatidylserine monolayer, whereas the polyamines and other monovalent cations did not increase the surface tension of the membrane appreciably. All cations used affected the vesicle mobility roughly in the order of the number of their valencies and linearly with respect to the logarithm of their concentrations of ions; vesicle surface charge densities are reduced by adsorption and screening of the counter ions depending on their valencies and concentrations. The degree of aggregation of lipid vesicles parallels somewhat that of the reduction of vesicle electrophoretic mobilities. However, the degree of membrane fusion induced by these cations parallels that of the increase in surface tension of the membranes induced by these cations.

Calcium-induced interaction of phospholipid vesicles and bilayer lipid membranes.

The interaction of unilamellar phospholipid vesicles with bilayer lipid membranes has been studied by observing the electrical conductance of the planar membrane. The presence of phosphatidylcholine vesicles as well as phosphatidylcholine/phosphatidylserine (1 : 1) vesicles on one side compartment of the bilayer membrane, but not phosphatidylserine vesicles, causes discrete fluctuations in the phosphatidylserine membrane conductance, which is also increased by at least an order of magnitude. These events are dependent on vesicle concentration as well as the presence of Ca2+. The results are interpreted in terms of the incorporation of domains of phosphatidylcholine into the membrane, which confer a higher conductance state.

Divalent cation-induced interaction of phospholipid vesicle and monolayer membranes.

The effects of phospholipid vesicles and divalent cations in the subphase solution on the surface tension of phospholipid monolayer membranes were studied in order to elucidate the nature of the divalent cation-induced vesicle membrane interaction. The monolayers were formed at the air/water interface. Various concentrations of unilamellar phospholipid (phosphatidylserine, phosphatidylcholine and their mixtures) vesicles and divalent cations (Mg2+, Ca2+, Mn2+, etc.) were introduced into the subphase solution of the monolayers. The changes of surface tension of monolayers were measured by the Wilhelmy plate (Teflon) method with respect to divalent ion concentrations and time. When a monolayer of phosphatidylserine and vesicles of phosphatidylserine/phosphatidylcholine (1 : 1) were used, there were critical concentrations of divalent cations to produce a large reduction in surface tension of the monolayer. These concentrations were 16 mM for Mg2+, 7 mM for Sr2+, 6 mM for Ca2+, 3.5 mM for Ba2+ and 1.8 mM for Mn2+. On the other hand, for a phosphatidylcholine monolayer and phosphatidylcholine vesicles, there was no change in surface tension of the monolayer up to 25 mM of any divalent ion used. When a phosphatidylserine monolayer and phosphatidylcholine vesicles were used, the order of divalent ions to effect the large reduction of surface tension was Mn2+ greater than Ca2+ greater than Mg2+ and their critical concentrations were in upon vesicle concentrations as well as the area/molecule of monolayers. For phosphatidylserine monolayers and phosphatidylserine/phosphatidylcholine : 1) vesicles, above the critical concentrations of Mn2+ and Ca2+, the surface tension decreased to a value close to the equilibrium pressure of the monolayers within 0.5 h. This decrease in surface tension of the monolayers is interpreted partly as the consequence of fusion of the vesicles with the monolayer membranes. The order and magnitude of divalent cation concentrations at which phosphatidylserine/phosphatidylcholine (1 : 1) and phosphatidylserine vesicle suspensions showed a large increase in turbidity were similar to those obtained in the above mentioned experiments.

Surface potential of phosphatidylserine monolayers. II. Divalent and monovalent ion binding.

Ion binding constants for phosphatidylserine membranes have been derived from the variation of the surface potential of phosphatidylserine monolayers with divalent cation concentrations in the presence of various monovalent salts in the aqueous subphase. The observed surface potential data for the monolayers, analyzed by use of the Gouy-Chapman diffuse potential theory, together with a simple binding reaction formula, yield, for Ca2+, Mg2+, Na+ and (Me)4N+ binding constant values of 30 M-1, 10 M-1, 0.6 M-1 and 0.05 M-1, respectively. The effect of pH on surface potential of phosphatidylserine monolayers was found to be dependent upon ionic species other than H+ in the subphase solution. The distinction between apparent and intrinsic dissociation constants of H+ for biomolecules was made in terms of ion binding due to other ions at the same site as for H+ in biomolecules.

Fusion of small unilamellar liposomes with phospholipid planar bilayer membranes and large single-bilayer vesicles.

Small unilamellar phosphatidylserine/phosphatidylcholine liposomes incubated on one side of planar phosphatidylserine bilayer membranes induced fluctuations and a sharp increase in the membrane conductance when the Ca2+ concentration was increased to a threshold of 3--5 mM in 100 mM NaCl, pH 7.4. Under the same ionic conditions, these liposomes fused with large (0.2 micrometer diameter) single-bilayer phosphatidylserine vesicles, as shown by a fluorescence assay for the mixing of internal aqueous contents of the two vesicle populations. The conductance behavior of the planar membranes was interpreted to be a consequence of the structural rearrangement of phospholipids during individual fusion events and the incorporation of domains of phosphatidylcholine into the Ca2+-complexed phosphatidylserine membrane. The small vesicles did not aggregate or fuse with one another at these Ca2+ concentrations, but fused preferentially with the phosphatidylserine membrane, analogous to simple exocytosis in biological membranes. Phosphatidylserine vesicles containing gramicidin A as a probe interacted with the planar membranes upon raising the Ca2+ concentration from 0.9 to 1.2 mM, as detected by an abrupt increase in the membrane conductance. In parallel experiments, these vesicles were shown to fuse with the large phosphatidylserine liposomes at the same Ca2+ concentration.

Surface potential of phosphatidylserine monolayers. I. Divalent ion binding effect.

Surface potentials of phosphatidylserine monolayers have been measured in the presence of different divalent ion concentrations in order to determine the way in which divalent ions bind to the membrane surface. The association constants for divalent ions (Mg2+, Ca2+ and Mn2+) with the phosphatidylserine membrane have been obtained from the experimental data and simple ion binding theory. The order of divalent ion binding to the membrane is Mn2+ greater than Ca2+ greater than Mg2+. However, none of the divalent ions used completely neutralized the negative charge of phosphatidylserine even at relatively high concentrations. The amounts of the divalent ions bound depended upon the concentration of the monovalent ions present in the subphase. It is suggested that the amounts of bound ions obtained from the use of radioisotope tracer methods may include a considerable contribution from the excess free ions in the double layer region of the phosphatidylserine membrane.

Divalent cation-induced phosphatidic acid membrane fusion. Effect of ion binding and membrane surface tension.

A study was made on the correlation between the degree of membrane fusion and surface tension increase of phosphatidic acid membranes caused by divalent cations. Membrane fusion was followed by the Tb3+/dipicolinic acid assay, monitoring the fluorescent intensity for mixing of the internal aqueous contents of small unilamellar lipid vesicles. The surface tension and surface potential of monolayers made of the same lipids as used in the fusion experiments were measured as a function of divalent cation concentration. It was found that the 'threshold' concentration to induce massive vesicle membrane fusion was the same for Ca2+ and Mg2+, and that the surface tension increase in the monolayer, induced by changing divalent cation concentration from zero to a concentration which corresponds to its threshold value, inducing vesicle membrane fusion, was approximately the same: 6.3 dyn/cm for both Ca2+ and Mg2+. Both the divalent cation's threshold concentrations as well as the surface tension change corresponding to the threshold concentration for the phosphatidic acid membrane were smaller than those for the phosphatidylserine membrane. The different fusion capability of these divalent cations for phosphatidic acid and phosphatidylserine membranes is discussed in terms of the different ion binding capabilities of these ions to the membranes.

Effect of glycosaminoglycans and PEG on fusion of Sendai virus with phosphatidylserine vesicles.

The fusion of Sendai virus with phosphatidylserine vesicles was monitored by a pyrene-phosphatidylcholine fluorescence assay. A strong influence of pH and ionic strength on the extent of fusion was observed. The negatively-charged polymers (dextran sulfate, heparin and chondroitin sulfate) inhibited the ability of the viruses to fuse with the liposomes. The extent of inhibition, for a given amount (w/v) of the polymers, was the greatest for dextran sulfate followed by heparin and chondroitin sulfate. The extent of inhibition depended on the pH and ionic strength of the solution; the lower the pH of the solution, the more effective the fusion inhibition by the polymers. The molecular weight of dextran sulfate (DS) influenced the inhibition effect, i.e., DS with higher molecular weight exhibited a stronger inhibition effect. The presence of sodium sulfate, even in excess concentration, had no inhibitory effect on fusion. On the other hand, PEG had an opposite effect on fusion compared to the negatively-charged polymers, and it decreased their inhibition effect when both were present in the same media. It is concluded that the inhibition of the fusion activity of Sendai virus results from the adherence of negatively-charged polymers to the virus surface preventing close contacts between the virus and liposome surface.

Association of lysozyme to phospholipid surfaces and vesicle fusion.

Lysozyme-induced fusion of phosphatidylserine (PS) vesicles was studied as a function of pH. Fusion, monitored by lipid-mixing, was measured by following the dilution of pyrene-labelled phosphatidylcholine, incorporated in PS vesicles, into unlabelled bilayers. It is demonstrated that lysozyme-induced fusion is pH-dependent and significant fusion is triggered at pH 5 or below. The interaction of lysozyme with the vesicle bilayer was characterized by measuring resonance energy transfer from tryptophane, present in the protein, to pyrene. It is shown that concomitant with fusion, a strong resonance energy transfer signal appears at pH 5 or below. Furthermore, in monolayer experiments it was found that addition of lysozyme to the subphase caused an increase in surface pressure, when the pH was kept below 5.5. Very low concentrations of lysozyme sufficed to bring about the observed effects. The results are taken to indicate that lysozyme-induced fusion results from penetration of protein into the hydrophobic core of the bilayer, occurring at acidic pH.

Isolation and characterization of large (0.5 - 1.0 micron) cytoskeleton-free vesicles from human and rabbit erythrocytes.

Large (0.5 - 1.0 micron) cytoskeleton-free vesicles were obtained, by 'budding', from fresh human and rabbit erythrocytes incubated at 45 degrees C and titrated with EDTA and CaCl2. This process occurs without hemolysis. The isolated vesicles maintain their cytoplasmic integrity and normal membrane orientation, and are resistant to hemolysis over the pH range 5.0 - 11.0 and temperature range 4-50 degrees C. The only membrane proteins detected in vesicles from human erythrocytes were band 3 region polypeptides and bands PAS-1, PAS-2 and PAS-3. Vesicles obtained from rabbit erythrocytes were similarly simple. Because of their size and stability these vesicles are amenable to both kinetic and quantitative analysis using the same experimental techniques employed in studies of synthetic lipid membranes. The results obtained in this study indicate that these vesicles are essentially markedly simplified biological cells, and thus may be useful as a biologically relevant model membrane system for examining the molecular interactions which occur within, across and between cell membranes.

Permeability of axon membranes to local anesthetics.

The permeability of the neutral form of tertiary amine local anesthetics across squid axon membranes was studied by utilizing three different experimental methods: (1) narcotic action of axon excitability was measured by monitoring the time derivative of action potential and the results were analyzed in terms of a diffusion reaction equation of local anesthetics to obtain their permeabilities; (2) the influx of local anesthetic into the axon was measured by use of the radioisotope tracer technique; and (3) the desorption rates of the neutral form of local anesthetics from lipid monolayers were measured and the desorption rate was correlated with permeability. The relative permeabilities obtained for procaine, lidocaine and tetracaine by the above three methods were comparable. The order of relative permeabilities was procaine greater than lidocaine greater than tetracaine, and had an inverse correlation with the partition coefficients of anesthetics at oil/water phases. Some discussion concerning the concept of permeability is made when the partition coefficient of a permeant molecule is high.