Insulin rapidly increases diacylglycerol by activating de novo phosphatidic acid synthesis.

The mechanisms whereby insulin increases diacylglycerol in BC3H-1 myocytes were examined. When [3H]arachidonate labeling of phospholipids was used as an indicator of phospholipase C activation, transient increases in [3H]diacylglycerol were observed between 0.5 and 10 minutes after the onset of insulin treatment. With [3H]glycerol labeling as an indicator of de novo phospholipid synthesis, [3H]diacylglycerol was increased maximally at 1 minute and remained elevated for 20 minutes. [3H]Glycerol-labeled diacylglycerol was largely derived directly from phosphatidic acid. Insulin increased de novo phosphatidic acid synthesis within 5 to 10 seconds; within 1 minute, this synthesis was 60 times greater than that of controls. Thus, the initial increase in diacylglycerol is due to both increased hydrolysis of phospholipids and a burst of de novo phosphatidic acid synthesis. After 5 to 10 minutes, de novo phosphatidic acid synthesis continues as a major source of diacylglycerol. Both phospholipid effects of insulin seem important for generating diacylglycerol and other phospholipid-derived intracellular signaling substances.

Selectivity of dihydropyridines for cardiac L-type and sympathetic N-type Ca2+ channels.

The blocking effects of cilnidipine and other dihydropyridines on L-type cardiac Ca2+ channels (I(Ca,L)) and N-type sympathetic Ca2+ channel currents (I(Ca,N)) were studied using a whole-cell patch-clamp technique. At -80 mV, cilnidipine had little inhibitory effect below concentrations of 1 microM on I(Ca,L) (IC50 value; 17 microM). However, 1 microM cilnidipine strongly shifted the steady-state inactivation curve of I(Ca,L) toward negative potentials without changing the current-voltage relationship. Each action of cilnidipine was characterized by a high affinity for the inactivated channel in preference to the resting channel. The IC50 values of dihydropyridines for I(Ca,L) were in the range between 0.01 and 10 microM, and those for I(Ca,N) were between 3 and 30 microM. Cilnidipine had the strongest affinity for I(Ca,N) among the dihydropyridines tested. These results suggest that cilnidipine did not cause hypotension-evoked tachycardia deficiency by depression of cardiac L-type channels but by sympathetic N-type channels blockade.

Effects of a dual L/N-type Ca(2+) channel blocker cilnidipine on neurally mediated chronotropic response in anesthetized dogs.

We investigated the effects of an L-type and N-type Ca(2+) channel blocker, cilnidipine, on neurally mediated chronotropic responses to clarify the anti-autonomic profile of cilnidipine in anesthetized dogs. Pretreatment with cilnidipine (0.3, 1.0 and 3.0 microg/kg, i.v.), which decreased mean blood pressure by 5 to 31 mm Hg, inhibited the changes in heart rate and plasma norepinephrine concentration induced by bilateral carotid artery occlusion, whereas it had no effect on vagal nerve stimulation-induced bradycardia. These results suggest that antihypertensive and antisympathetic doses of cilnidipine fail to influence chronotropic responses mediated by parasympathetic nerve activation in the in vivo canine heart.

Hemodialysis effect on serum boron level in the patients with long term hemodialysis.

Serum and dialysate boron levels in 17 patients with long term hemodialysis (HD) were determined by inductively coupled plasma emission spectrometry (ICPES). Serum boron level was compared with the value of age matched 467 healthy controls and the relationship between serum and dialysate boron level was analyzed. The results showed that serum boron level was significantly higher at the beginning of HD, and lower at the completion of HD in comparison with controls. Although the dialysate was contaminated with trace boron, HD resulted in an excessive decrease of serum boron, rather than boron exposure from the dialysate. Boron hemodialyzability was almost proportional to the gradient of the boron level at the beginning of HD and it could be controlled by the adjustment of the gradient. In conclusion, the serum boron level was very much disturbed in long term HD patients. If boron excess in serum at the beginning of HD, or deficiency at the completion of HD may contribute to the complications of HD patients, fine adjustment and close surveillance of the gradient should be taken into account.

Quality control of diphtheria tetanus acellular pertussis combined (DTaP) vaccines in Japan.

Diphtheria tetanus acellular pertussis combined (DTaP) vaccines have been successfully used in Japan by controlling their potencies and toxicities with animal models. In accordance with the recent practical introduction of DTaP vaccines of various formulations, a question has been raised in other nations as to the efficacy of a quality control system based on animal tests and standard preparations. The World Health Organization issued its guidelines on the production and quality control of acellular pertussis vaccines in 1998 along with the concept of quality control by ensuring that production lots were consistent with clinical trial lots, rather than by comparing them with standard preparations in traditional laboratory tests. However, because it is not feasible to evaluate the combined use of vaccines from different manufacturers in a clinical study, the alternative trend of quality control may give rise to a difficulty in rationalizing the practical immunizations to use vaccines of different brands in a mixed consequence. A standardized national regulation system to ensure the equivalence of approved products would be essential for such an immunization practice. The success of the Japanese DTaP vaccination suggests the possibility of an effective quality control of DTaP vaccines by means of standardized test systems.

A clinical study on SSP (silver spike point) electro-therapy combined with splint therapy for temporo-mandibular joint dysfunction.

When the functional limits of the muscles related to the temporo-mandibular joint and adjacent tissue exceed their anatomical capability, pain, crepitation, and functional abnormality appear as the main complaints. Although the precise nature of the condition is unknown, pain at the temporo-mandibular joint sometimes in combination with muscular tension is assumed to be due to compression of the myoneural mechanism. It is reported that occlusal lifting using a splint enables the alleviation of this muscular tension. On the other hand, there are only a few reports on the usefulness of SSP therapy for Temporo-Mandibular Joint Dysfunction. We studied the efficacy of SSP therapy combined with splint therapy in 33 patients diagnosed as having Temporo-Mandibular Joint Dysfunction who consulted our department primarily due to pain, and report our findings below. Evaluation of the results was conducted 2 weeks later. Very beneficial results were seen in 6 cases. Beneficial results were seen in 7 cases. Slightly beneficial results were seen in 18 cases, while there were no changes found in 2 cases. When combined SSP and splint therapies were conducted for Temporo-Mandibular Joint Dysfunction, favorable results were seen in about 90% of the cases.

Genotypes and infection sites in an outbreak of multidrug-resistant Pseudomonas aeruginosa.

An outbreak of multidrug-resistant (MDR) Pseudomonas aeruginosa occurred in an acute care hospital in Japan, which lasted for more than three years. During January 2006 to June 2009, 59 hospitalised patients with MDR P. aeruginosa were mainly detected by urine culture in the first half, whereas isolation from respiratory tract samples became dominant in the latter half of the outbreak. Non-duplicate MDR P. aeruginosa isolates were available from 51 patients and all isolates were positive for bla(VIM-2). Pulsed-field gel electrophoresis (PFGE) analysis categorised the isolates into three major clusters; types A, B and C with eight, 19 and 21 isolates, respectively. The outbreak started with patients harbouring PFGE type A strains, followed by type B, and type C strains. Multivariate analysis demonstrated that patients with PFGE type C strains were more likely to be detected by respiratory tract samples (odds ratio: 11.87; 95% confidence interval: 1.21-116.86). Improved aseptic urethral catheter care controlled PFGE type A and type B strains and improvement in respiratory care procedures finally contained the transmission of PFGE type C strains.

Stimulation of Bordetella pertussis adenylate cyclase toxin intoxication by its hemolysin domain.

The internalization of the N-terminal catalytic domain of Bordetella pertussis adenylate cyclase toxin (ACT) across the cytoplasmic membrane has been considered to occur independently from protein-protein interactions which can lead to oligomerization required for hemolytic activity by its C-terminal hemolysin domain. Here we report that when added in excess, this hemolysin domain stimulates the internalization, suggesting the involvement of protein-protein interactions in cell-invasive activity of ACT, as well as its hemolytic activity.

Suppression of platelet aggregation by Bordetella pertussis adenylate cyclase toxin.

The effect of Bordetella pertussis adenylate cyclase toxin (ACT) on platelet aggregation was investigated. This cell-invasive adenylate cyclase completely suppressed ADP (10 microM)-induced aggregation of rabbit platelets at 3 micrograms/ml and strongly suppressed thrombin (0. 2 U/ml)-induced aggregation at 10 micrograms/ml. The suppression was accompanied by marked increase in platelet intracellular cyclic AMP (cAMP) content and was diminished by the anti-ACT monoclonal antibody B7E11. A catalytically inactive point mutant of ACT did not show the suppressive effect. Since an increase of cAMP content is a known cause of platelet dysfunction, these results indicate that the observed platelet inactivation was due to the catalytic activity of ACT through increase of intracellular cAMP.

IFN-gamma-mediated protection against intracerebral challenge with Bordetella pertussis in mice.

Mice inoculated with whole cell pertussis vaccine (WCV) acquired protection against intracerebral challenge with Bordetella pertussis without any appreciable antibody production against pertussis toxin or filamentous hemagglutinin. Spleen cells from mice immunized with WCV produced a significant amount of IFN-gamma upon stimulation in vitro with WCV. Furthermore, mice inoculated with recombinant IFN-gamma along with a suboptimal dose of WCV survived longer than those that received WCV alone. These results suggest that, in WCV-immune mice, IFN-gamma plays an important role in protection against intracerebral challenge with B. pertussis.

Post-transfusion hepatitis type B: long incubation period and poor prognosis in compromised hosts.

Seven cases of post-transfusion hepatitis type B [PTH(B)] were investigated. PTH(B) developed in 4 patients more than 65 years old and in 4 patients after treatment of a malignant disease (2 cases of gastric cancer and one each of ovarian cancer and chronic myelogenous leukemia, respectively). The mean incubation period was 78 days (70-90) in patients with non-malignant diseases and 147 days (105-200) in patients with malignant diseases. The symptoms of acute hepatic failure developed in 6 patients and 5 of them expired. One fatal case revealed 4 units of blood and an investigation of 4 donors revealed that one of them was an HBsAg carrier with negative serum HBsAg by the reverse passive hemagglutination (RPHA) method. From these results, it was concluded that compromised hosts such as aged patients or patients with malignant diseases are apt to contract severe PTH(B) with a long incubation period when the transfused blood contains small amounts of HBV.

Nitric oxide induction by pertussis toxin in mouse spleen cells via gamma interferon.

We examined the major pathogenic substances of Bordetella pertussis for the ability to induce nitric oxide, and important biological function of macrophages, via gamma interferon in spleen cells. B. pertussis, which produces a variety of pathogenic substances, including pertussis toxin and filamentous hemagglutinin, causes a severe respiratory disease. Nitric oxide was detected in the culture fluid of spleen cells stimulated with pertussis toxin or its B oligomer but not in the culture fluid of spleen cells stimulated with the A protomer of pertussis toxin or with filamentous hemagglutinin. Incubation of the peritoneal exudate macrophages with pertussis toxin, B oligomer, A protomer, or filamentous hemagglutinin induced little nitric oxide, whereas incubation with gamma interferon induced a significant amount of nitric oxide. The induction of nitric oxide in spleen cells stimulated with pertussis toxin was completely inhibited by anti-gamma interferon antibody. The treatment of spleen cells with anti-Thy-1.2 antibody plus complement followed by stimulation with pertussis toxin decreased the secretion of gamma interferon and nitric oxide. These results suggest that gamma interferon from T lymphocytes stimulated with pertussis toxin induces nitric oxide.

Use of antiserum-coated latex particles for serotyping Streptococcus pneumoniae.

The Quellung reaction provides a standard means for serotyping Streptococcus pneumoniae, but it requires microscopic examination with skillful technique. We have developed an improved agglutination method with anti-rabbit IgG-coated latex particles, which are sensitized with pooled antisera for serotyping/serogrouping S. pneumoniae. Our method is as specific and sensitive as the Quellung test, and much easier to perform.

Insulin increases membrane and cytosolic protein kinase C activity in BC3H-1 myocytes.

Insulin treatment stimulated the activity of the Ca2+- and phospholipid-dependent protein kinase (protein kinase C) in both cytosolic and membrane fractions of BC3H-1 myocytes. Within 60 s of insulin treatment, membrane protein kinase C activity increased 2-fold, diminished toward control levels transiently, and then increased 2-fold again after 15 min. Cytosolic protein kinase C activity increased more gradually and steadily up to 80% over a 20-min period. Increases in protein kinase C activity were dose-dependent and were not simply a result of translocation of cytosolic enzyme (although this may have occurred), as total activity was also increased. The increase in protein kinase C activity was not inhibited by cycloheximide (which also increased protein kinase C activity and 2-deoxyglucose transport) and was still evident following anion exchange chromatography. The insulin effect was decidedly different from those of 12-O-tetradecanoylphorbol-13-acetate and phenylephrine using histone III-S as substrate. Phenylephrine decreased cytosolic protein kinase C activity while increasing membrane activity; 12-O-tetradecanoylphorbol-13-acetate only decreased cytosolic protein kinase C activity. The early insulin-induced increases in membrane protein kinase C activity may be related to increased diacylglycerol generation from de novo phosphatidic acid synthesis, as there were rapid increases in [3H]glycerol incorporation into diacylglycerol, and transient increases in phospholipid hydrolysis, as there were transient rapid increases in [3H]diacylglycerol in cells prelabeled with [3H]arachidonate. Later, sustained increases in membrane and cytosolic protein kinase C activity may reflect the continuous activation of de novo phospholipid synthesis, as there were associated increases in [3H]glycerol incorporation into diacylglycerol at later, as well as very early time points.

Insulin provokes co-ordinated increases in the synthesis of phosphatidylinositol, phosphatidylinositol phosphates and the phosphatidylinositol-glycan in BC3H-1 myocytes.

BC3H-1 myocytes were cultured in the presence of [3H]inositol or [3H]glucosamine during their entire growth cycle to ensure that all lipids containing inositol and glucosamine were labelled to isotopic equilibrium or maximal specific radioactivity. After such labelling, a lipid (or group of lipids), which was labelled with both inositol and glucosamine, was observed to migrate between phosphatidylinositol 4-phosphate and phosphatidylinositol (PI) in two different t.l.c. systems. Insulin provoked rapid, sizeable, increases in the inositol-labelling of this lipid (presumably a PI-glycan), and these increases were similar to those observed in PI and PI phosphates. Our results indicate that insulin provokes co-ordinated increases in the net synthesis de novo of PI and its derivatives, PI phosphates and the PI-glycan, in BC3H-1 myocytes. This increase in synthesis of PI may serve as the mechanism for replenishing the PI-glycan during stimulation of its hydrolysis by insulin. Moreover, increases in the content of the PI-glycan may contribute to increases in the generation of head-group 'mediators' during insulin action.

Hepatitis B virus antigen and antibodies in alcoholics. Etiological role of HBV in liver diseases of alcoholic patients.

A study was carried out to clarify the pathogenetic role of HBV in alcoholic patients with liver diseases. The incidence of serological markers of HBV infection was investigated in these patients and the histological characteristics were compared among the alcoholic patients with and without HBV markers. A high percentage of patients were positive for either HBsAg, anti-HBs or anti-HBc. In six of eight patients with positive HBsAg, liver histology showed viral features, but in 23 of 39 patients with positive anti-HBs and/or low titre anti-HBc and in 12 of 18 patients with negative HBV markers liver histology showed alcoholic features. From these results it is concluded that HBV presumably plays a major role in the pathogenesis of liver disease in alcoholic patients with persistent HBV infection but not in patients with positive antibodies.