Simultaneous assay of activated platelet count and platelet-activating capacity by P-selectin detection using K2-EDTA-treated whole blood for antiplatelet agents.

INTRODUCTION: It is well recognized that examinations of activated platelets (aPLTs) and platelet-activating capacity are very important to observe and prevent embolic diseases (events) such as ischemic stroke and myocardial infarction. Previously, we reported an appropriate measurement technique of aPLT for clinical assay. In this paper, we investigated stable conditions for measurement of activating capacity of platelets. METHODS: Blood samples were taken from healthy volunteers using anticoagulants of 2K-EDTA, sodium citrate and heparin, and platelets were stimulated with adenosine diphosphate (ADP) or collagen. We demonstrated platelet-activating capacity by detection of scattering light, absorbance, microscopic observation, and P-selectin (CD62P) expression. We also performed basic experiments in seven healthy volunteers to test the clinical application of these assays with monitoring aspirin therapy. RESULTS: We judged that samples of whole blood with 2K-EDTA were suitable for CD62P expression assay as functional assessments of platelet activity, because platelets treated with anticoagulants such as sodium citrate and heparin were extremely damaged after stimulation, and it was difficult to measure the CD62P expression by flow cytometry. For optimal results, samples should be tested within 1 h after the drawing of blood and stimulated with ADP or collagen for 10 min. The CD62P-positive platelet value of blood from volunteers who had taken aspirin was decreased, and platelet activation was inhibited as well. CONCLUSION: The simultaneous assay of aPLT and platelet-activating capacity by CD62P detection using whole blood treated with the K2-EDTA anticoagulant was useful for the monitoring of antiplatelet drugs.

Treatment with a Ca(2+) channel blocker, barnidipine, reduces platelet-derived growth factor B-chain mRNA in glomeruli of spontaneously hypertensive rats.

We investigated the effect of barnidipine hydrochloride, a Ca(2+) channel blocker, on the glomerular level of mRNA expression of platelet-derived growth factor (PDGF) B-chain and transforming growth factor (TGF)-beta(1) in spontaneously hypertensive rats (SHR) with reverse transcription and polymerase chain reaction. Thirteen-week-old SHR were provided with food containing barnidipine (0.6 mg/g of food, average dose during treatment: 53 mg/kg of body mass/day) for 3 weeks. A stable reduction in systolic blood pressure relative to that of age-matched control SHR was recorded after week 1 of therapy. Although no renal histological changes were observed after 3 weeks of treatment with barnidipine, the level of expression of PDGF B-chain mRNA in glomeruli was significantly reduced relative to that in control SHR. The glomerular level of TGF-beta(1) mRNA expression was not affected by the treatment. Treatment with barnidipine significantly reduced the excretion of urinary protein. Thus, the stable reduction in systemic blood pressure by barnidipine is associated with a reduction in PDGF B-chain mRNA expression in the glomerulus and reduction in urinary protein excretion in SHR.

Effect of hyperthermia on cyclin B expression in a human glioblastoma cell line.

Effects of hyperthermia on the cell kinetics of glioblastoma cells were investigated using flow cytometry. Pulse-labeling with 5-bromodeoxyuridine (BUdR) and chasing of the labeled cells revealed temporary accumulation of the labeled cells in G2M phase and a reduction of DNA synthesis. The level of cyclin B rises rapidly in G2 phase and falls at the end of mitosis in normal cycling cells. Cyclin B binds to p34cdc2, resulting in histone kinase activity which is necessary for the initiation of mitosis. The amount of p34cdc2 remains constant throughout the cell cycle. The level of cyclin B was measured using an anti-cyclin B antibody and flow cytometry in order to investigate the cause of the G2 accumulation induced by hyperthermia. A low level of cyclin B, in comparison with that of normal cycling cells, persisted for more than 3 h after hyperthermia. These results indicate that the temporary accumulation of cells in G2M phase after hyperthermia may be caused, at least in part, by an insufficient level of cyclin B.

Chemical structure-activity of cnidium rhizome-derived phthalides for the competence inhibition of proliferation in primary cultures of mouse aorta smooth muscle cells.

Inhibitory effects of cnidium rhizome-derived phthalides on competence and progression phases of fetal bovine serum (10%)-induced proliferation were compared in primary cultures of mouse aorta smooth muscle cells (SMC). Their potencies for the competence inhibition were in the order of senkyunolide L ((Z)-6-hydroxy-7-chloro-6,7-dihydroligustilide) > senkyunolide H ((Z)-6,7-dihydroxy-6,7-dihydroligustilide) > senkyunolide J ((3S)-(E)-6,7-dihydroxy-3,6,7,8-tetrahydroligustilide) > senkyunolide I ((E)-6,7-dihydroxy-6,7-dihydroligustilide) > ligustilide = senkyunolide A ((3S)-3,8-dihydroligustilide) > butylidenephthalide. The order of their potencies for the progression inhibition was parallel with that for the competence inhibition. Senkyunolide L is considered to have been formed during the extraction of cnidium. These results demonstrate that the (Z)-6,7-dihydroxy isomer of the dihydroligustilide derivatives is essential for the anti-competent effect on proliferation of the SMC in primary culture. Senkyunolide H in cnidium rhizome may be a prototype for a new anti-atherosclerotic drug.

Antiproliferative effects of the traditional Chinese medicine shimotsu-to, its component cnidium rhizome and derived compounds on primary cultures of mouse aorta smooth muscle cells.

Antiproliferative effects of the Japanese-Sino medicine Shimotsu-to (a combined prescription of cnidium rhizome, angelica root, peony root and rehmannia root) were investigated in the primary culture of smooth muscle cells (SMC) of mouse aorta. Fetal bovine serum (10%)-induced proliferation of primary cultured SMC was inhibited by Shimotsu-to at 4, 20, 100 or 500 micrograms/ml. The inhibitory effect was selective on SMC and due to cnidium rhizome or angelica root. The IC50 values of senkyunolide H, senkyunolide A, ligustilide and butylidenephthalide derived from cnidium were below 0.1, 1.52, 1.68 and 3.25 micrograms/ml, respectively. These results indicate that the antiproliferative effect of Shimotsu-to may depend on these cnidium-derived phthalides.

[Nutritional support in critically ill patients; with special reference to fat emulsion and carnitine].

Energy metabolism of fat emulsion was studied in rats with septic condition. The results indicate that fat emulsion was rapidly eliminated from the blood stream and metabolized readily even in such condition. Based on these findings, we have actively employed fat emulsion clinically as an energy source of septic patients. In septic rats, it was demonstrated that levels of carnitine decreased and that this decrease was based upon a decrease of synthesis. When carnitine was administered together with fat emulsion, the energy metabolism returned to approximately normal level. This reports also describes the absorption and utilization of orally administered fat in septic rats.

Side effects in the treatment of herpetic keratitis.

Various side effects due to antiherpetic drugs observed in the last ten years in our department were studied. A total of 132 patients were treated with 5-iodo-2'-deoxyuridine (IDU), 69 with trifluorothymidine (F3T), 58 with acyclovir (ACV) and 33 with adenine arabinoside (ara-A). Patch tests were routinely done when patients exhibited contact dermatitis. Of the patients treated with IDU, 3 (2.3%) showed contact dermatitis, 2 (1.5%) follicular conjunctivitis and 1 (0.8%) punctate keratopathy. Of the patients treated with F3T, 7 (10.1%) exhibited contact dermatitis and 1 (1.4%) follicular conjunctivitis. In the group treated with ACV, 2 (3.4%) patients showed punctate keratopathy. The patients who received ara-A did not show any side effects. We found that F3T caused contact dermatitis more frequently in Japanese people than Europeans. These side effects were resolved by switching to another anti-herpetic drug without the occurrence of cross-allergy. Therefore, switching to another drug is strongly recommended when patients exhibit side effects in the treatment of herpetic keratitis. Other complications were allergy to atropine and to drug preservative.

Thalamic control of spontaneous alpha-rhythm and evoked responses.

EEG, depth recordings from the thalamus, visual-evoked potentials, and readiness motor potentials were analyzed with a computer in 40 patients with involuntary movements and intractable pain who underwent stereotactic thalamotomy. It was demonstrated that there were significant correlations in alpha-rhythms between the ventrolateral (VL) nucleus and the frontocentral cortex, and also between the centromedian nucleus and the centroparietal cortex, with the phase difference of the cortex lagging behind the thalamus. Relationships between the thalamic VL nucleus and the frontocentral cortex were significant not only in alpha-rhythms, but also in evoked responses following photic stimulations, voluntary movements, and electrical thalamic stimulation. Thalamotomy produced a bilaterally decreased synchronization of the cortical alpha-rhythms, which recovered within 6 months.

VL thalamotomy for the treatment of tremor in patients with thalamic syndrome.

A 59-year-old female who underwent successful thalamotomy for tremor in thalamic syndrome was reported. Thalamic syndrome including tremor of the left limbs and slight dysesthesia over the left side of the face was thought to be due to the vascular lesion located in the right thalamus and adjacent structure by detailed radiological and electrophysiological examination. Significant alleviation of tremor was achieved by right VL thalamotomy without any complication.