[Results of treatment of out-of-hospital cardiopulmonary arrest patients transferred to the UOEH hospital].

We have accepted patients suffering out-of-hospital cardiopulmonary arrest (CPA) since September 1997. In this study, we retrospectively reviewed the etiology of CPA and the outcome of the patients. We also investigated the effects of the pre-hospital cardiopulmonary resuscitation (CPR) by a bystander or emergency life-saving technicians (ELST) on the restoration of spontaneous circulation. Seventy-seven patients were transferred to our hospital from September 1997 to April 2000, 10 of who underwent bystander (basic) CPR. All the patients underwent advanced CPR by the attending physician immediately after admission to the emergency room. The etiology of CPA was categorized into three groups: (1) unknown endogenous disease (43 patients, 56%), (2) exacerbation of previous disease such as heart, respiratory or cerebrovascular disease (12 patients, 15%), (3) accident or suicide (22 patients, 29%). Spontaneous circulation of 30 patients was restored temporary, but only two patients recovered fully and were discharged from the hospital. The rate of restored spontaneous circulation of the CPA patients with bystander CPR was higher compared to that of the CPA patients without bystander CPR. The two fully recovered patients underwent bystander CPR. The CPR by ELST did not affect the resuscitated rate significantly. These results indicate that the most common etiology of CPA is unknown endogenous disease, and the rate of full recovery is very low (2.6%). This study suggests that bystander CPR may contribute to an improved outcome of the patients suffering out-of-hospital CPA.

The severity of hyperdynamic circulation may predict the effects of direct hemoperfusion with the adsorbent column using polymyxin B-immobilized fiber in patients with gram-negative septic shock.

It has been reported that direct hemoperfusion with the adsorbent column using polymyxin B-immobilized fiber (DHP with PMX-F column) ameliorates hyperdynamic circulation in septic shock and improves survival rate. However, the clinical characteristics of patients with an improvement of septic shock after DHP with PMX-F column have not been evaluated. To clarify this issue, the clinical profiles of 46 patients who were suggested to have gram-negative septic shock and treated using DHP with PMX-F column were analyzed retrospectively. Of 46 patients, 31 were diagnosed with gram-negative septic shock (G group). Mean arterial pressure (MAP) just before DHP with PMX-F column was not different between the G and the non-G group. As compared with the non-G group, the G group had a higher cardiac index (CI) and a lower systemic vascular resistance (SVR). Significant increases in MAP and SVR with a significant decrease in CI were observed after DHP with PMX-F column in the G group. In the non-G group, MAP was significantly increased after the DHP therapy, but systemic hemodynamics were unchanged. Patients in the G group who fulfilled the following criteria were considered as the effective group: MAP was elevated more than 10 mm Hg or 125% of the basal MAP and/or the dose of vasopressors was reduced after DHP with PMX-F column. Twenty-one patients (67.8%) were in the effective group. In comparison with the effective group, the noneffective group was characterized by a significant increase in CI before DHP with PMX-F column. All patients with a CI less than 6 L/min/m2 were in the effective group. These data suggest that DHP with PMX-F column was useful for patients with gram-negative septic shock who did not have severe hyperdynamic circulation.

[Occupational physicians' concern and fact-finding methods regarding cardiopulmonary resuscitation education for employees in the workplace].

The fact-finding methods and occupational health physicians' concern regarding cardiopulmonary resuscitation (CPR) education for employees in the workplace was investigated. Questionnaires were sent to 138 occupational health physicians who graduated from the University of Occupational and Environmental Health, Japan, during the past 10 years. Eighty-two physicians replied to the questionnaire (response rate: 59%). Of these, 98% answered that it was necessary to educate employees regarding CPR, but only 32% of the physicians were actually training employees in CPR. Of the physicians who have had experience educating employees, 85% said that they had been requested by the company to do so. The major reason for not educating employees regarding CPR was that there had been no request from the company. The percentage of physicians who have performed CPR in the workplace was 35%. There was no relation between the request for CPR for employees from the company and whether the practice was actually being done in the workplace. Of the physicians who have not as yet taught CPR to employees, 70% affirmed that they would like to do so in the future. We concluded that companies needed to be enlightened regarding the necessity of CPR education for employees.

Voltage-dependent ionic currents in dissociated paratracheal ganglion cells of the rat.

1. Conventional whole-cell voltage-clamp technique was used to study the electrophysiological and pharmacological properties of voltage-dependent Na+, K+ and Ca2+ channels in parasympathetic neurones enzymatically dissociated from the paratracheal ganglia of rat trachea. The voltage-dependent Na+, K+ and Ca2+ currents (INa, IK and ICa) were separated by the use of ion subtraction and pharmacological treatments. 2. INa was activated by a step depolarization more positive than -50 mV and fully activated at positive potentials more than +10 mV. The inactivation phase of INa consisted of fast and slow exponential components (tau if and tau is, respectively). The tau if and tau is were voltage dependent and decreased with a more positive step pulse. 3. The time course for recovery of INa from the complete inactivation exhibited two exponential processes. 4. The reversal potential of INa was equal to the Na+ equilibrium potential (ENa) and resembled a simple Na+ electrode depending only on external Na+ concentration. 5. Tetrodotoxin (TTX) reduced INa without affecting the current kinetics in a concentration-dependent manner, and the concentration of half-maximal inhibition (IC50) was 6 x 10(-9) M. There was no TTX-resistant component of INa in any of the cells tested. 6. Scorpion toxin increased the peak amplitude of INa and prolonged the inactivation phase in a time- and concentration-dependent manner. In the presence of toxin, both tau is and the fractional contribution of the slow current component to total INa increased concentration dependently. 7. High-threshold (L-type) ICa was activated by a step depolarization more positive than -30 mV and reached a peak at near 0 mV in the external solution with 2.5 mM Ca2+. The current was inactivated to only a small extent (< 10%) during 100 ms of depolarizing step pulse. There was no low-threshold (T-type) ICa in this preparation. 8. The maximum ICa in individual current-voltage (I-V) relationships was saturated by an increase in extracellular Ca2+ concentration ([Ca2+]o). The I-V relationships were also shifted along the voltage axis to the more positive potential with increasing [Ca2+]o. 9. The inactivation process of the L-type ICa was dependent on Ca2+ influxes (ICa-dependent inactivation). 10. Relative maximum peak currents of divalent cations passing through the L-type Ca2+ channels were in the order of IBa > ICa > ISr. 11. Organic and inorganic Ca2+ antagonists blocked the ICa in a concentration-dependent manner.(ABSTRACT TRUNCATED AT 400 WORDS)

gamma-Aminobutyric acid-induced response in rat dissociated paratracheal ganglion cells.

1. The pharmacologic properties of gamma-aminobutyric acid (GABA)-induced Cl- current (ICl) were studied in the paratracheal ganglion cells freshly dissociated from 7- to 10-day-old rat trachea in a whole-cell recording mode by the use of a conventional patch-clamp technique. 2. GABA- and muscimol-induced currents increased sigmoidally in a concentration-dependent manner, and both currents reversed at approximately -3 mV, which was close to the Cl- equilibrium potential (ECl). 3. Strychnine (STR) at low concentration and bicuculline (BIC) inhibited GABA response competitively, whereas STR at the higher concentrations, benzylpenicillin (PCG), or picrotoxin (PTX) inhibited noncompetitively. Inhibition of GABA response by PCG but not other antagonists was voltage dependent, indicating that PCG acts as a Cl- channel blocker. 4. The concentration-response curve of pentobarbital sodium (PB)-induced ICl was bell shaped. At concentrations higher than 10(-3) M, both the peak and plateau currents decreased, and a transient "hump" current appeared immediately after washing out PB. In the presence of PB, the concentration-response curve of GABA shifted toward left without changing the maximum response. 5. Although diazepam (DZP) at concentration used did not induce a response, it potentiated the GABA response in a concentration-dependent manner between 10(-8) and 10(-6) M. DZP also caused a parallel shift toward left in the concentration-response curve of GABA. 6. PB or DZP further enhanced the GABA response in the presence of the other agent. 7. It is concluded that the properties of GABAA receptors in the paratracheal ganglion cells are essentially similar to those reported in other preparations.

Direct modulation of GABAA receptor by intracellular ATP in dissociated nucleus tractus solitarii neurones of rat.

1. Effect of intracellular ATP on Cl- current (ICl) mediated by the GABA (gamma-aminobutyric acid) receptor subtype, GABAA, was studied in dissociated nucleus tractus solitarii (NTS) neurones using the whole-cell mode of patch clamp. A concentration-jump technique termed 'Y tube' was used to rapidly apply agents externally. Dissociated neurones were obtained from 1- to 3-week-old rats. 2. When the patch-pipette solution contained 2 mM-ATP, the amplitude of ICl elicited by 10(-5) M-GABA did not show any time-dependent decrease (apparent run-down), for more than 60 min after the initial recording. In the presence of ATP, the half-maximum concentration (KD) and Hill coefficient calculated from the GABA concentration-response curve were 9.12 microM and 1.47, respectively. 3. In the absence of intracellular ATP, the amplitude of GABA-induced ICl decreased with time. The relative peak amplitudes after 20 and 60 min from the initial recording were 0.40 +/- 0.09 (n = 11) and 0.16 +/- 0.05 (n = 8) with respect to the initial response. 4. Removal of Mg2+ from the internal solution induced run-down of the GABA response even in the presence of 2 mM-intracellular ATP, suggesting that both intracellular ATP (2 mM or more) and Mg2+ are necessary to prevent run-down of the GABA response. 5. Activation of dephosphorylation processes by alkaline phosphatase (100-200 microM) did not affect the GABA response in neurones perfused with internal solution containing 2 mM-ATP and 3 mM-Mg2+. Blocking the dephosphorylation process by okadaic acid, a phosphatase inhibitor, did not prevent the run-down of the GABA response. 6. Calcium influxes passing through both the voltage-dependent L-type Ca2+ channel and the glutamate receptor-operated cation channel did not affect ICl induced by GABA. 7. GABA-induced ICl was also maintained by adding 2 mM-ADP or ATP gamma S (adenosine-5'-O-3-thiotriphosphate) to the internal solution containing Mg2+. Addition of 2 mM-adenosine, AMP, cyclic AMP, AMP-PNP (adenylimido-diphosphate) or ADP beta S (adenosine-5'-O-2-thiodiphosphate) to the internal solution did not prevent the run-down of the GABA response even in the presence of 3 mM-intracellular Mg2+. Based on the chemical specificity of these ATP analogues, it is suggested that there is an ATP-sensitive binding site (ATP receptor) in the cytoplasmic side of the cell membrane.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholine-activated ionic currents in isolated paratracheal ganglion cells of the rat.

The electrophysiological property of acetylcholine (ACh)-induced current (IACh) was studied in paratracheal ganglion cells freshly isolated from rat trachea under whole-cell voltage-clamp condition. IACh consisted of an initial transient peak component and a successive steady-state plateau one. The peak component increased in a sigmoidal fashion with increasing ACh concentration. The IACh was mimicked by nicotine. The current-voltage relationship for the IACh showed inward rectification at the positive membrane potentials beyond the reversal potential (EACh). In a K(+)-free solution, the EACh was close to the Na+ equilibrium potential. The IACh was blocked by either D-tubocurarine or atropine. The ion selectivity of ACh-activated channels to various monovalent cations was weak, and similar to those of other preparations. It was concluded that the IACh in rat paratracheal ganglion cells was mediated by nicotinic receptor activation.

Strychnine-induced potassium current in isolated dorsal root ganglion cells of the rat.

1. Effects of strychnine (Str) on the dissociated dorsal root ganglion (DRG) cells of the rat have been investigated in whole-cells configuration by a conventional patch-clamp technique. 2. gamma-Aminobutyric acid (GABA)-induced Cl- current (ICl) increased sigmoidally with increasing concentration. The half-maximal response (Ka) was 3 x 10(-5) M and the Hill coefficient was 1.5. Both Str and bicuculline inhibited the GABA-induced ICl in a concentration-dependent manner. 3. Str itself could elicit the current at concentrations over 10(-5) M, at which concentrations the GABA response was completely suppressed. The concentration-response curve for the Str-induced current was bell-shaped, and a nearly maximum response occurred at 3 x 10(-4) M. A transient 'hump' current appeared immediately after the wash-out of external solution containing high concentrations of Str over 3 x 10(-4) M. 4. The Str-induced outward current and a transient 'hump' current were augmented by the removal of extracellular K+ and were suppressed by the substitution of intracellular K+ for Cs+. But the current was not sensitive to extracellular Na+, Ca2+ and Cl-. 5. The reversal potential of Str-induced current (EStr) was -75 mV, which was close to the K+ equilibrium potential (EK = -76.3 mV). The change of EStr for a ten fold change in extracellular K+ concentration was 58 mV, indicating that the membrane behaves like a K+ electrode in the presence of Str. The reversal potential of the 'hump' current was also close to EK. 6. The Str-induced outward current was antagonized by K+ channel blockers such as Ba2+, tetraethylammonium (TEA)-chloride, and 4-aminopyridine (4-AP) in a concentration-dependent manner. 7. The Str-induced K+ current was not affected by internal perfusion of bis(gamma-aminophenoxy)ethane-N, N,N',N-tetraacetic acid (BAPTA), indicating that the Str response does not result in the activation of K+ conductance by the intracellular Ca2+.

Oral immunization with recombinant Streptococcus lactis carrying the Streptococcus mutans surface protein antigen gene.

A recombinant Streptococcus lactis strain which carries the structural gene for a surface protein antigen (PAc) of 190,000 daltons from Streptococcus mutans serotype c was constructed for development of an oral vaccine against dental caries. The gene from S. mutans MT8148 joined to shuttle vector pSA3 was successfully transformed into S. lactis IL1403. A small amount of PAc was detected in the cell homogenate and cytoplasmic fraction of the recombinant S. lactis, but not in the culture supernatant of the recombinant, by Western immunoblotting and dot immunoblotting. The level of PAc-specific mRNA in the recombinant strain was lower than that in S. mutans MT8148. However, significant salivary immunoglobulin A and serum immunoglobulin G responses to PAc were induced in mice immunized orally with the recombinant S. lactis.

Cytotoxic effects of aflatoxin B1 and its association with cellular components in chicken embryo primary cultured cells.

We have examined the cytotoxicity and cellular incorporation of aflatoxin B1 (AFB1) in several types of established and primary cultured cells. The inhibition of DNA synthesis by AFB1 at 1 microgram/ml was about 0-30% in the established cell lines, including human hepatic cells. In chicken primary hepatocytes, however, DNA synthesis as well as RNA and protein syntheses were strongly inhibited by much lower concentrations of AFB1, e.g., 0.1 microgram/ml. In contrast, chicken primary fibroblasts showed almost no significant response to the toxin. Microsomal cytochrome P-450 activities in hepatic tissues were 10-20-fold higher than those in fibroblastic tissues. The amount of [3H]AFB1 incorporated into acid-insoluble materials in the primary hepatocytes was also 10-100-fold more than that in the primary fibroblasts. However, a significant amount of AFB1, which was enough to induce cytotoxic effects on the primary hepatocytes, could be incorporated into the primary fibroblasts when the concentrations of AFB1 were increased. Characterization of the AFB1-associated cellular components showed that most of them were DNA, RNA, and proteins in the primary hepatocytes, while in the primary fibroblasts a large portion of the incorporated AFB1 was recovered from lipid fractions. In addition, the selective binding of [3H]AFB1 to several proteins was observed only in the primary hepatocytes. The possible role of the AFB1-binding proteins are also discussed.

Lipofuscin and lipofuscin-like substances.

Lipofuscin is defined as being a yellowish brown, lipid-rich, heterogeneous, cytoplasmic granular pigment emitting an intense yellow autofluorescence when excited with ultraviolet light, which accumulates in various tissues of animals during their aging. It is believed that the pigments are derived from the reaction of some of reactive secondary products including malonaldehyde, formed during membranous lipid peroxidation, with amino groups of phospholipids and proteins, etc., and that these formations are accompanied by alteration of the membrane structure and inactivation of the enzymes. The fluorescence measurement of the pigments is widely used as a parameter of lipid peroxidation in vivo as well as in vitro. However, their origin, chemical structure, biological significance or fate has not as yet been fully elucidated. This article introduces and discusses the recent studies on these problems.

Conformation between the substrate-binding site and heme of cytochrome P-450 studied by excitation energy transfer.

The conformation between the substrate-binding site and heme of cytochrome P-450 was studied by excitation energy transfer. Cytochrome P-450 was obtained from the hepatic microsomes of polychlorinated biphenyl-treated male rats, and ten polycyclic aromatic hydrocarbons were used as substrates. The energy transfer from the substrate to the heme of the enzyme was measured according to the Förster equation. On the basis of the assumption that the substrates are bound at different positions in the plane of the same substrate-binding site, the position of the heme in relation to the substrate-binding site was determined in solution and in the presence of synthetic phospholipid. The results demonstrated that the distance between the substrate-binding site and the heme of cytochrome P-450 was greater when the enzyme was incorporated into micelles of phospholipid than when in solution, and that the conformational relationship of the substrate-binding site towards the heme was changed by an angle of 21 degrees on incorporation of the enzyme into phospholipid micelles.

Immunopotentiative effect of polysaccharide from kefir grain, KGF-C, administered orally in mice.

Since a water-soluble polysaccharide (KGF-C) from the kefir grains was shown to have the property of retarding tumor growth in vivo when administered orally, the effect of KGF-C was examined on antibody responses to thymus-dependent antigen, sheep red blood cells (SRBC), and thymus-independent antigen, dinitrophenyl-Ficoll and trinitrophenyl-lipopolysaccharide. Antibody response in mice intubated with KGF-C was enhanced to low doses of SRBC, but not to optimal or high doses. The optimal dose of KGF-C required for the enhancement was 100 mg/kg body weight. The time-course studies on KGF-C administration implied that KGF-C exerted its effect on the early events of anti-SRBC response. The enhancement was not due to the alteration of kinetics of anti-SRBC responses. Furthermore, the enhancing effect on antibody responses to thymus-independent antigens, such as dinitrophenyl-Ficoll and trinitrophenyl-lipopolysaccharide, was observed neither in nu/nu nor in nu/+ mice, and the effect on delayed-type hypersensitivity response to a low dose of SRBC in normal mice was also found. These findings suggest that the oral immune enhancement by KGF-C is elucidated probably through T-cell but not through B-cell participation.

Conformational change of cytochrome P-450 indicated by the measurement of fluorescence-energy transfer.

The conformation of cytochrome P-450 was studied in relation to the interaction with its substrate by the measurement of fluorescence energy transfer. Cytochrome P-450 I-c and cytochrome P-450 II-d, both obtained from the hepatic microsome of polychlorinated biphenyl-treated rats, were used as P-450 type and P-448 type, respectively, and benzo[a]pyrene and 7-ethoxycoumarin were used as substrate. The distance between the donor and acceptor, which was described by the Förster equation, was calculated on the basis of the fluorescence-energy transfer between the substrate as a donor and the heme of the enzyme as an acceptor. The distance was apparently changed by incorporation of the enzyme into phospholipid or by reduction of the heme, indicating that the treatments changed the conformation of the enzyme. Differences in the conformational change were observed between the two enzymes, and the conformational change of cytochrome P-450 II-d using benzo[a]pyrene as a substrate was different from that using 7-ethoxycoumarin. The results suggest that the substrate-binding sites of the two enzymes differ in position toward the heme, and that there are at least two different substrate sites in cytochrome P-450 II-d, one for benzo[a]pyrene and another for 7-ethoxycoumarin.

Fluorescent substances in mouse and human sera as a parameter of in vivo lipid peroxidation.

Water-soluble fluorescent substances like lipofuscin were found in the protein fraction of mouse and human sera and had fluorescence characteristics with excitation and emission maxima at 335-340 and 435-440 nm for mice, and at 355-360 and 430-435 nm for human, respectively. When oxidized [U-14 C]linoleic acid was given intraperitoneally to mice, or added to the serum in vitro, both the fluorescence intensity and radioactivity of serum protein increased dose-dependently in the two tests. Also, the fluorescent substances responded well to acceleration of in vivo lipid peroxidation caused by carbon tetrachloride. These results indicate that the substances were some binding compounds between the degraded lipids and serum proteins, and that they could be taken as a parameter of in vivo lipid peroxidation. The distribution of the pigments in the serum proteins, albumin and globulins, was shown to depend upon the number of free amino groups in each protein which appear to be binding sites of degraded lipids. Spectral characteristics and some chemical properties of the substances suggest that they might not be conjugated Schiff bases formed from protein and malondialdehyde but might be due to some other stable compounds. Significantly high levels of the substances were observed in sera of patients with diabetes and hypertension.

Elevation of thiobarbituric acid values in the rat liver intoxicated by T-2 toxin.

In the present study we first demonstrated that T-2 toxin markedly stimulated lipid peroxidation specifically in the liver of rats. The amount of lipid peroxides in the liver, estimated by the thiobarbituric acid (TBA) method, increased dose dependently, being proportional to the extent of its acute toxicity measured by various parameters in rats fed a commercial diet. Further, to elucidate the mechanism of lipid peroxidation and its role in hepatic injury caused by T-2 toxin, time-course studies on the correlation between lipid peroxide content and some biological and histopathological data were undertaken in rats given 4 mg of the toxin/kg perorally. The TBA reactive substances in the liver began to increase after 6 hr. However, much earlier than this there were some other alterations, which included decreases in the amount of cytochrome P-450 in the liver, of GPT (thereafter an increase) and phospholipids in the plasma, and of basophilic masses in the hepatocytes (arrayed as a rough endoplasmic reticulum in the electron micrograph). The vitamin E-deficient study showed that vitamin E markedly inhibited the stimulative effect of T-2 toxin on lipid peroxidation, but not diminish any other measured parameters of the injury. The toxin-induced stimulation of lipid peroxidation does not appear to be caused by activation of microsomal NADPH-cytochrome c reductase nor by a decrease in the level of cytosolic glutathione peroxidase. These results suggest that T-2 toxin might induce some alteration of the membrane structure and consequently might stimulate lipid peroxidation in situ.

Interactions of aflatoxin B1 and blood components of various species in vitro: interconversion of aflatoxin B1 and aflatoxicol in the blood.

The fate of aflatoxin B1 (AFB1) in the blood of various species of animals was studied in vitro. Examination of the distribution of radioactivity in blood incubated with [14C]AFB1 at 37 degrees C showed that high levels of radioactivity were associated with blood cells. The radioactivity was readily removed from the blood cells by washing with fresh plasma, indicating loose binding of AFB1 to blood cells. Most of the radioactivity in plasma was bound to protein. These results suggest that a large part of the AFB1 in blood in vivo may be carried not only by the plasma proteins but also by the blood cells. When chloroform extracts of plasma of [14C]AFB1-treated mouse, rat, duckling, and hamster blood were developed by thin-layer chromatography, high levels of radioactivity were found in both the AFB1 region and the aflatoxicol (AFL) region. Incubation of blood with nonradioactive AFB1 and AFL showed marked interconversion of AFB1 and AFL in the blood of rats, hamsters, mice, and Mongolian gerbils, but not in the blood of guinea pigs, rhesus monkeys, squirrel monkeys, or humans. Interconversion occurred in red blood cell suspensions but not in plasma, indicating that the red blood cells are responsible for AFB1-AFL interconversion in the blood.

Effect of orally administered polysaccharide from kefir grain on delayed-type hypersensitivity and tumor growth in mice.

The effect of oral administration of a polysaccharide (KGF-C), isolated from the kefir grain, on delayed-type hypersensitivity (DTH) induced by picryl chloride and on the growth of solid tumor was examined in mice. KGF-C caused an increase in DTH response in intact mice and also tumor-bearing mice. The growth of tumor inoculated after the DTH test was markedly inhibited in the groups with high DTH response. A significant correlation between the DTH response and the antitumor activity was observed in intact mice.