Biomarkers for the evaluation of immunological properties during the shikoku walking pilgrimage.

It is important to determine the immunological properties for the maintenance of health. We chose the Shikoku Walking Pilgrimage to assess the proper biomarkers for the evaluation of immunological properties. We examined whether the Shikoku Walking Pilgrimage could have a positive effect on the mental and physical health of walking participants by using several biomarkers proposed by our laboratory. Twelve non-randomized healthy male volunteers including 3 twice attendees walked the Shikoku Walking Pilgrimage distance of 58.9 km over 3 days. Plasma, serum, urine, and saliva were collected from the volunteers during the pilgrimage and at 1 week before and after it. Immunological biomarkers, including lipid metabolism, oxidative stress, immune function, and catecholamines, were measured. Additionally, mood state scores, alertness, autonomic nervous system activity, and body motion levels during sleep were assessed. A significant decrease was observed in the subjective tension-anxiety levels and in the concentrations of serum low-density lipoprotein cholesterol, plasma hydroxyoctadecadienoic acid (HODE), and urine adrenaline during the pilgrimage as compared to the values of these parameters before the participants embarked on the pilgrimage. The serum levels of brain-derived neurotrophic factor (BDNF) were significantly increased 1 week after the pilgrimage relative to those assessed previously. No significant differences in subjective fatigue and the flicker perception threshold were observed. These results suggest that the Shikoku Walking Pilgrimage can exert a positive effect on mental and physical health as particularly shown in the reduction of tensionanxiety and oxidative stress without the accompaniment of fatigue. HODE correlated significantly with typical immunological marker natural killer cell activity and immunoglobulin G. This suggests that there are promising biomarkers such as HODE, NK activity, BDNF, LDL-c, and IgG for assessing the immunological properties.

Development of Algorithm for Discriminating Hydrometeor Particle Types with a Synergistic Use of CloudSat and CALIPSO.

We developed a method for classifying hydrometeor particle types, including cloud and precipitation phase and ice crystal habit, by a synergistic use of CloudSat/Cloud Profiling Radar (CPR) and Cloud-Aerosol Lidar and Infrared Pathfinder Satellite Observations (CALIPSO)/Cloud-Aerosol LIdar with Orthogonal Polarization (CALIOP). We investigated how the cloud phase and ice crystal habit characterized by CALIOP globally relate with radar reflectivity and temperature. The global relationship thus identified was employed to develop an algorithm for hydrometeor type classification with CPR alone. The CPR-based type classification was then combined with CALIPSO-based type characterization to give CPR-CALIOP synergy classification. A unique aspect of this algorithm is to exploit and combine the lidar's sensitivity to thin ice clouds and the radar's ability to penetrate light precipitation to offer more complete picture of vertically resolved hydrometeor type classification than has been provided by previous studies. Given the complementary nature of radar and lidar detections of hydrometeors, our algorithm delivers thirteen hydrometeor types: warm water, supercooled water, randomly-oriented ice crystal (3D-ice), horizontally-oriented plate (2D-plate), 3D-ice+2D-plate, liquid drizzle, mixed-phase drizzle, rain, snow, mixed-phase cloud, water+liquid drizzle, water+rain and unknown. The global statistics of three-dimensional occurrence frequency of each hydrometeor type revealed that 3D-ice contributes the most to the total cloud occurrence frequency (53.8%), followed by supercooled water (14.3%), 2D-plate (9.2%), rain (5.9%), warm water (5.7%), snow (4.8%), mixed-phase drizzle (2.3%), and the remaining types (4.0%). This hydrometeor type classification provides useful observation-based information for climate model diagnostics in representation of cloud phase and their microphysical characteristics.

Comparison of the conformational stability of the molten globule and native states of horse cytochrome c. Effects of acetylation, heat, urea and guanidine-hydrochloride.

The molten globule state has been assumed to be a major intermediate of protein folding. We compared the stability of the native and acidic molten globule states of horse ferricytochrome c against heat, urea and guanidine hydrochloride (Gdn-HCl) using the intact species and species modified by various degrees of acetylation of the lysyl epsilon-amino groups. After acetylation, the amino groups cannot protonate at acidic pH. Thermal and urea-induced unfolding transitions measured by far-UV circular dichroism and differential scanning calorimetry showed that, whereas acetylation stabilizes the molten globule state at pH 2, it destabilizes the native state at pH 7, suggesting a difference in their mechanisms of conformational stability. On the other hand, the effects of Gdn-Hcl were remarkable. Contrary to what was expected from the thermal and urea-induced unfolding transitions, the Gdn-HCl-induced unfolding transition of the native state at pH 7 was insensitive to the extent of acetylation. At pH 2, Gdn-HCl at low concentrations stabilized the molten globule state and, at high concentrations, destabilized it. Consideration of the difference in the effects of Gdn-HCl from those of urea or heat indicated that, whereas the net positive charge repulsion destabilizes the molten globule state at pH 2, the local negative charge repulsion produced by acetylation of amino groups, and not the net charge, critically destabilizes the native state at pH 7. These results predict that, because of its ionic nature, Gdn-HCl will produce substantially different effects on the conformational states of some proteins compared with those of urea.

Molten globule of cytochrome c studied by small angle X-ray scattering.

Recent advances in the small angle X-ray scattering technique have made it possible to obtain information on the spatial distribution of protein structure in solution, such as the radius of gyration and shape of the molecule, in a relatively short time. We have studied the various conformational states of horse ferricytochrome c by small angle X-ray scattering. Whereas Guinier plots showed that the radius of gyration of the molten globule state is slightly larger than that of the native state, Kratky plots indicated that their shape is globular. To clarify the mechanism of the formation of the molten globule state, we measured, by the small angle X-ray scattering, the equilibrium transition at pH 2 from the acid-unfolded state to the molten globule state, which was induced either by the addition of NaCl or the acetylation of lysyl amino groups. The Kratky plots at various stages of folding showed an isoscattering point, strongly suggesting a two-state mechanism. For both the NaCl and acetylation-induced transitions, the transition curve determined from Kratky plots agreed well with the transition curves from far-ultraviolet circular dichroism, tryptophan fluorescence, and Soret absorption, further supporting the two-state mechanism.

Guanidine hydrochloride-induced folding of proteins.

Guanidine hydrochloride (Gdn-HCl) is the most commonly used denaturant for proteins. Contrary to expectation, we found that Gdn-HCl at low concentrations refolds acid-unfolded apomyoglobin and cytochrome c, stabilizing the molten globule state, i.e. a compact denatured state with a significant amount of secondary structure, but substantially disordered tertiary structure. A further increase in Gdn-HCl concentration, above 1 M, caused co-operative unfolding of the molten globule state. Similar sequential folding and unfolding transitions were also observed at neutral pH with a synthetic amphiphilic peptide consisting of Lys and Leu residues, indicating the generality of the phenomenon. Although the Gdn-HCl-induced refolding and unfolding transitions were puzzling at first glance, we show that they are readily interpreted in terms of the differential action of Gdn-HCl. We also show that the comparison of the unfolding curves for the molten globule and native states provides a measure of the buried surface area upon formation of the molten globule state.

Identification of the phospholipid-binding site of human beta(2)-glycoprotein I domain V by heteronuclear magnetic resonance.

To understand the mechanism of the interaction between human beta(2)-glycoprotein I (beta(2)-GPI) and negatively charged phospholipids, we determined the three-dimensional solution structure of the fifth domain of beta(2)-GPI by heteronuclear multidimensional NMR. The results showed that the molecule is composed of well-defined four anti-parallel beta-strands and two short alpha-helices, as well as a long highly flexible loop. Backbone dynamic analysis demonstrated significant mobility of the flexible loop on a subnanosecond time scale. Structural modeling of the nicked fifth domain, in which the Lys317-Thr318 peptide bond was specifically cleaved, revealed the importance of this long C-terminal loop for the interaction between beta(2)-GPI and negatively charged phospholipids. A titration experiment with the anionic surfactant SDS showed that this highly mobile loop, as well as the short beta-hairpin between betaC and betaD strands, which is rich in positively charged residues, specifically interact with the surfactant. The mobile loop, together with the surrounding positively charged residues, probably construct the binding site for negatively charged phospholipids such as cardiolipin.

Dose-dependent doxycycline-mediated adrenocorticotropic hormone secretion from encapsulated Tet-on proopiomelanocortin Neuro2A cells in the subarachnoid space.

We previously reported that polymer-encapsulated mouse neuroblastoma cells that are capable of secreting beta-endorphin may reduce pain sensitivity in rats after capsule implantation into the cerebrospinal fluid (CSF)-filled subarachnoid space of the spinal cord. The neuroblastoma cells carry the proopiomelanocortin (POMC) gene that encodes the precursor of adrenocorticotropic hormone (ACTH) and beta-endorphin. To control the expression of these hormones in the present study, a promoter that is inducible by administration of tetracycline derivatives such as doxycycline (Dox) was linked to the POMC gene. Encapsulated cells in the CSF space of rats stimulated by four intraperitoneal doses of Dox responded with ACTH expression as determined in a subsequence 36-hr in vitro incubation. The amount of ACTH released was dependent on the in vivo Dox dose. These findings indicate that gene expression in xenogeneic cells in the CSF space can be manipulated by injection of a relatively innocuous drug, and suggest that this system may be applicable to cell transplantation therapy in patients with central nervous system diseases that require temporary control of ligand delivery.

Thermal unfolding of tetrameric melittin: comparison with the molten globule state of cytochrome c.

Whereas melittin at micromolar concentrations is unfolded under conditions of low salt at neutral pH, it transforms to a tetrameric alpha-helical structure under several conditions, such as high peptide concentration, high anion concentration, or alkaline pH. The anion- and pH-dependent stabilization of the tetrameric structure is similar to that of the molten globule state of several acid-denatured proteins, suggesting that tetrameric melittin might be a state similar to the molten globule state. To test this possibility, we studied the thermal unfolding of tetrameric melittin using far-UV CD and differential scanning calorimetry. The latter technique revealed a broad but distinct heat absorption peak. The heat absorption curves were consistent with the unfolding transition observed by CD and were explainable by a 2-state transition mechanism between the tetrameric alpha-helical state and the monomeric unfolded state. From the peptide or salt-concentration dependence of unfolding, the heat capacity change upon unfolding was estimated to be 5 kJ (mol of tetramer)-1 K-1 at 30 degrees C and decreased with increasing temperature. The observed change in heat capacity was much smaller than that predicted from the crystallographic structure (9.2 kJ (mol of tetramer)-1 K-1), suggesting that the hydrophobic residues of tetrameric melittin in solution are exposed in comparison with the crystallographic structure. However, the results also indicate that the structure is more ordered than that of a typical molten globule state. We consider that the conformation is intermediate between the molten globule state and the native state of globular proteins.

Trifluoroethanol-induced conformational transition of hen egg-white lysozyme studied by small-angle X-ray scattering.

The trifluoroethanol (TFE)-induced conformational transition of hen lysozyme was studied with the combined use of far-UV circular dichroism (CD) and small-angle X-ray scattering. At pH 2.0 and 20 degrees C, the addition of TFE to the native lysozyme induced a cooperative transition to an intermediate state with an increased helical content (TFE state). Small-angle X-ray scattering measurements indicated that the TFE state has a radius of gyration which is 20% larger than that of the native state and assumes a chain-like conformation with some remaining globularity. The TFE-induced transition curves obtained by CD and the small-angle X-ray scattering measurements agreed well, consistent with a two-state transition mechanism. A singular value decomposition analysis of Kratky plots of the small-angle X-ray scattering profiles indicated that two basic scattering functions reproduce the observed spectra, further confirming the validity of a two-state approximation.

Role of the N- and C-terminal domains of bovine beta 2-glycoprotein I in its interaction with cardiolipin.

beta 2-Glycoprotein I (beta 2-GPI) is a cofactor in the recognition of the phospholipid antigen cardiolipin by anti-cardiolipin antibodies in autoimmune diseases such as systemic lupus erythematosus. We examined the interactions of various forms of bovine beta 2-GPI, such as its intact form, desialylated form (Asialo-beta 2-GPI), N-terminal domain (Domain I), and modified forms of beta 2-GPI and Asialo-beta 2-GPI with nicks in their C-terminal domains, with phospholipid liposomes under different conditions of pH and ionic strength. We found that at neutral pH and low ionic strength, beta 2-GPI became bound to liposome membranes containing cardiolipin, phosphatidylglycerol, phosphatidylserine, phosphatidylserine, phosphatidic acid, or phosphatidylinositol, but not phosphatidylcholine alone. The number of phospholipids involved in the binding seemed to depend on the head group structure of the negatively charged phospholipids, but the dissociation constant did not, being about 10(-8) M, except that for the interaction with phosphatidylinositol, which was one order of magnitude lower. We also found that Domain I and Asialo-beta 2-GPI bound to liposome membranes containing negatively charged phospholipids, and that in the interaction with cardiolipin, their dissociation constants were about 10(-6) and 10(-8) M, respectively. At neutral pH and both low and high ionic strengths, the affinities of the nicked forms of beta 2-GPI and Asialo-beta 2-GPI for cardiolipin were both lower than those of their intact forms but similar to that of Domain I.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure and function of the recombinant fifth domain of human beta 2-glycoprotein I: effects of specific cleavage between Lys77 and Thr78.

In order to elucidate the mechanism of binding of beta 2-glycoprotein I (beta 2-GPI) to cardiolipin (CL), we constructed a high-level expression system for the C-terminal domain (Domain V) of beta 2-GPI using Pichia pastoris and studied its conformation and liposome-binding activity. Purified Domain V was found to have the native disulfide bonds. It had a compactly folded conformation, judging from the circular dichroism spectrum, and exhibited a cooperative unfolding transition induced by pH or urea. Also, it bound liposomes containing CL. Commercially available human beta 2-GPI is known to be selectively cleaved between Lys 317 and Thr 318. We found that bovine factor Xa weakly but specifically cleaves the corresponding site of recombinant Domain V, i.e., the peptide bond between Lys 77 and Thr 78. The conformation of the "nicked" Domain V, which was cleaved at this site, was examined by circular dichroism and fluorescence measurements, and concluded to be similar to that of the intact protein. The stability of the nicked Domain V to urea was slightly lower than that of the intact protein. Although both Domains V bound to liposomes containing CL, the affinity of the nicked Domain V was greatly reduced in comparison with the intact protein, indicating that the cleavage of the peptide bond between Lys 77 and Thr 78 controls the binding to CL. In addition, analysis of the fluorescence spectra in the presence and absence of CL liposomes indicated that Trp 76 is involved in the binding site. These results suggest that the region including Trp 76, Lys 77, and Thr 78 has a critical role in binding to CL.

Transplantation of xenogeneic cells secreting beta-endorphin for pain treatment: analysis of the ability of components of complement to penetrate through polymer capsules.

The permeation of component of complement and secreted peptides through polymer capsules (PM30, K6305, and K5708) were examined. To analyze permeability by complement, the degree of hemolysis of sensitized sheep erythrocytes (EA) (1 x 10(9)/ml) enclosed in each type of capsule was examined after 24-h incubation in culture medium containing 10% human serum. PM30 and K6305 prevented the permeation of complement well, while K5708 did not. EA suspended in alginate prevented hemolysis even in K5708. Peptide permeation through the capsules was assessed by measuring the concentration of ACTH secreted by proopiomelanocortin (POMC)-gene-transfected-Neuro2A in the culture medium on days 4, 7, 14, 21, and 28 after encapsulation. The ACTH levels in the culture medium remained high until day 28. Alginate appeared to prevent the secretion, because ACTH levels decreased in alginate-suspended cells after day 14. The PM30-K6306 double capsules containing cell lines, Neuro2A, BHK21 (hamster fibroblasts), L929 (mouse fibroblasts), and HF-SKFII (human fibroblasts) were transplanted into the cerebrospinal fluid (CSF) space of the monkeys in the lumber region. The morphological examination showed the partial survival of Neuro2A, and BHK21 and HF-SKFII, which were cells concordant with the monkeys. On the other hand, L929 cells, which were discordant with the monkeys, could not survive at all. Because these results suggest that the complement components penetrate the polymer capsules, concordant cells are preferable for xenografting with polymer capsules into the CSF space.

Long-term functional assessment of encapsulated cells transfected with Tet-On system.

In our previous study, xenogeneic mouse neuroblastoma cells bearing the POMC gene, the precursor of ACTH and beta-endorphin, were implanted within polymer capsules into the CSF space of rats. Although ACTH and beta-endorphin were secreted, we were not able to control the amounts or times of hormone release. A promoter that is inducible by administration of tetracycline derivatives (Tet) was linked to the POMC gene to control its gene expression (Neuro2A-Tet-On-POMC; NTP). The results showed that POMC gene expression in the implanted encapsulated NTP cells could be regulated in a dose-dependent manner by Tet administration to the hosts. However, no analysis of gene control with the Tet-On system over a long period has been performed. In this study, encapsulated NTP cells were treated in vitro with doxycycline (Dox) (1.0, 10, 100, 1000 ng/ml) continuously for a month. On day 4, the amount of ACTH secretion was dependent on the Dox dose. But in the course of the experiment, the difference of ACTH secretion among those treated with Dox 10, 100, and 1000 ng/ml was eliminated. On the other hand, NTP cells, which were treated with Dox (1000 ng/ml) just on days 7, 14, 21, and 28, secreted almost the same amount of ACTH in 24 h. From these results, for clinical use, an NTP cell line that secretes enough opiate to reduce pain sensitivity without Dox should be established, and Dox could then be administered if necessary.

Vascular permeability enhancing activity of Porphyromonas gingivalis protease in guinea pigs.

Porphyromonas gingivalis protease, which had been isolated from a culture supernatant, caused vascular permeability enhancement in a dose-dependent manner when injected into guinea pig skin. The permeability-enhancing reaction caused by the protease was not affected by treatment with antihistamine, but was greatly augmented by simultaneous injection of a kinin potentiator, carboxypeptidase N inhibitor. However, the reaction was inhibited by soybean trypsin inhibitor or alpha 2-antiplasmin, although both of these inhibitors could not inhibit P. gingivalis protease at all by themselves. A bradykinin-degrading enzyme, carboxypeptidase B, weakened this vascular reaction. Results described indicate that the permeability-enhancing reaction induced by the protease is caused by activation, of the kallikrein-kinin cascade in the tissue.

Activation of blood clotting factors by microbial proteinases.

There are very few reports on the involvement of bacterial proteinases on the blood clotting system using both human plasma and purified clotting factors. We studied whether microbial proteinases from the opportunistic pathogens Candida albicans, Pseudomonas aeruginosa and Serratia marcescens activate the blood clotting cascade by using normal human plasma, human plasmas deficient in clotting factor XII or X, and also by using purified clotting factors XII, X and prothrombin. All proteinases tested activated either clotting factor XII or prothrombin in vitro, thus resulting in generation of thrombin. Clotting factor X was converted to the active form (Xa) by both Candida and Pseudomonas proteinases, but not by Serratia proteinase. These results suggest that peripheral and systemic blood circulation may be impaired by activation of the blood clotting cascade by microbial infections, especially in septic patients, which would enhance disseminated intravascular coagulation and multi-organ failure.

Local cerebral glucose utilization in the postictal phase of amygdaloid kindled rats.

Local cerebral glucose utilization was measured by means of the quantitative autoradiographic 2-[14C]deoxyglucose method during the postictal phase of various seizure stages of amygdaloid kindling in conscious rats. The partially kindled animals exhibited a partial seizure such as chewing and/or head nodding, and the fully kindled animals, a generalized tonic-clonic convulsion. The control animals were implanted with an electrode, but not electrically stimulated. Cerebral glucose utilization of the fully kindled animals was deeply depressed in the postictal phase as compared to the control, and that of the partially kindled animals was moderately decreased. The side-to-side differences of cerebral glucose utilization were observed only in the partially kindled group in which glucose utilization was more depressed on the side of stimulation. Among the structures with depressed glucose utilization, only one structure, the interpeduncular nucleus, showed a relative increase in glucose utilization during the postictal phase of the kindled groups. As the postictal phase has been considered as a period of inhibition, these results may indicate that the neural networks linking the interpeduncular nucleus play an active role in the mechanisms of termination of a seizure and postictal refractoriness.

Influence of sympathetic nerves on development of responsiveness of the chick smooth muscle to drugs.

The role of sympathetic nerves in the development of responsiveness of smooth muscle to drugs was investigated using the expansor secondariorum muscle of 2- to 40-day-old chicks. The normal developmental decrease in the responsiveness of the muscle to acetylcholine was prevented by surgical transection of the nerve or chronic treatment with guanethidine, while it was facilitated by chronic treatment with dimethylphenylpiperazinium. Sensitivity of the muscle to noradrenaline, remaining constant during normal development, was increased by nerve section or guanethidine treatment, while it was slightly decreased by dimethylphenylpipe-azinium treatment. These results suggest that sympathetic nerves regulate the development of the responsiveness of the chick expansor secundariorum muscle to drugs, at least the developmental decrease in responsiveness to acetylcholine.

Degradation of human dentine collagen by an enzyme produced by the yeast Candida albicans.

An extracellular collagenolytic enzyme separated from a culture medium of this pathogenic yeast was found to attack undenatured predentine collagen as seen in scanning electron micrographs. After treatment with the enzyme at pH 4.0, but not by that acidity alone, dentine tubules were less easily distinguished and the collagen fibres were less well-organized.

[Change in the volume of extra cellular fluid in coronary artery bypass grafting (CABG); comparison between off-pump and on-pump CABG].

During the perioperative period for off-pump coronary artery bypass surgery (OPCAB) and on-pump coronary artery bypass surgery (on-pump CABG), the volume of extra cellular fluid (ECF) was measured. The subjects were elective adult coronary artery bypass surgery cases, consisting of 13 OPCAB cases and 7 on-pump CABG cases. The ECF volume was measured the day before surgery, immediately after surgery, and 2, 4, 6, 8, 12, 24 and 48 hours after surgery, with a bioimpedance analyzer (XITRON 4000 C). ECF volume variation was defined as the difference from the preoperative value divided by body weight, and was expressed in %BW. At the same time, respiratory-index and leukocyte count were measured. The maximum postoperative ECF volume was 3.13 +/- 2.6 %BW in the OPCAB group and 5.36 +/- 2.0 %BW in the on-pump CABG group, that is, significantly higher in the on-pump CABG group. The ECF volume started to increase in the on-pump CABG group immediately after surgery (4.38 +/- 1.8 %BW in the on-pump CABG group and 2.07 +/- 2.4 %BW in the OPCAB group), reaching its peak 6 hours after surgery in the on-pump CABG group and 4 hours after surgery in the OPCAB group. Thereafter, the volume gradually decreased, and 48 hours after surgery the volume decreased in the OPCAB group to 0.064 +/- 1.5 %BW, or to about the same value as the preoperative value, whereas in the on-pump CABG group the volume remained high: 1.9 +/- 2.9 % BW. There was no significant difference between the 2 groups in the change in respiratory-index. The leukocyte count remained significantly higher in the on-pump CABG group. The ECF volume was measured by the bioimpedance measuring method. This is a useful method of measuring the volume non-invasively and continuously. In the OPCAB group, the increase in postoperative ECF volume was less, and recovery to the preoperative level was faster than in the on-pump CABG group.

[Late results of coronary artery bypass grafting with the internal thoracic artery].

Between November 1981 and December 1991, 755 patients underwent isolated coronary artery bypass grafting. We compared postoperative events, graft patency and survival rates of the patients who received at least one internal thoracic artery graft (n = 517) with those of the patients who had only saphenous vein bypass grafts (n = 238). No significant difference was found between the 2 groups in patient's age at the operation, female ratio and the incidence of emergency operation. The operative mortality was not significantly different in either group. Graft patency was better for internal thoracic artery grafts than for SVG (ITA grafts 97.1%, SVG 85.6% p less than 0.0001). Graft-LAD patency rate was also better for ITA grafts than for SVG (ITA grafts 97.7%, SVG 86.8%, p less than 0.0001). The incidence of late cardiac events (late cardiac death, reoperation, myocardial infarction) is lower in patients with ITA. Furthermore, patients who received ITA grafts had a better survival rate at 8 years (91.1% versus 85.3%, p = 0.048) than those who had vein bypass grafts alone. We conclude that the use of ITA grafts enhances the long-term benefits including long-term survival rate. This is the first observation in Japanese population.