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Biomedical subjects

M Kaibara

Publications and source records attributed to M Kaibara.

At least 73 records · Page 4Linked to original sources

Reconstituted collagen is not capable of activating factor XII but causes intrinsic coagulation by activating platelets.

Using a rheological technique to measure the coagulation of plasma in collagen-coated tubes, we studied the intrinsic coagulant activities of different types (I, III, IV and V) and structures of reconstituted collagen. Recalcified, platelet-free plasma (PFP) in contact with the collagen surface did not clot, irrespective of the type and structure of collagen. Coagulation of platelet-rich plasma (PRP) did occur, although the time of onset of coagulation was highly dependent on the type and structure of the collagen used. Coagulation of PRP occurred rapidly on a collagen surface consisting of highly ordered fibrils (banded structure) to which large numbers of aggregated platelets adhered with shape change. In contrast, initiation of coagulation was delayed in PRP after incubation with 5 mM dibutyrylcyclic AMP (DB-cAMP) for 50 min. Coagulation of PRP was completely suppressed after 30 min incubation with colchicine (5.0 mg/ml). This suggested that disruption of platelet microtubules completely suppressed the stimulation of platelets associated with the initiation of intrinsic coagulation on the collagen surface. We conclude that reconstituted collagen is not capable of activating factor XII. Initiation of coagulation of recalcified PRP in contact with the reconstituted collagen surface is caused by the activation of platelets.

Adult↗

Inhibition of the muscarinic receptor-activated K+ current by N-ethylmaleimide in rabbit heart.

The effects of N-ethylmaleimide (NEM), a sulfhydryl alkylating agent, on the ACh-activated K+ current were examined in single cells from rabbit hearts using whole-cell and single channel patch clamp techniques. Bath application of NEM (50 microM) or the muscarinic antagonist, atropine (1 microM) completely inhibited the ACh-activated K+ current in whole-cell recordings. In cell-attached patch conditions, the inhibitory effect of NEM was still observed; in contrast, atropine was ineffective when the agents were bath applied, indicating that the site of action of NEM is different from that of atropine. Inside-out patch recordings confirmed that GTP was required on the internal side of the membrane for activation of this K+ channel when ACh was present in the pipette. NEM abolished this GTP-activated K+ channel activity. GTP gamma S, a non-hydrolysable GTP analogue, was able to activate this K+ channel in the absence of a muscarinic agonist, an effect thought to be due to the direct activation of GTP-binding proteins. Pretreatment with NEM almost completely prevented this effect of GTP gamma S. In contrast, after the activation of the K+ channel by GTP gamma S had reached a steady-state, NEM failed to show a significant inhibitory effect. These results demonstrate that NEM prevents the activation of muscarinic receptor-regulated K+ channel and suggest an involvement of alkylation of the GTP-binding proteins which are coupled to this type of K+ channel.

Acetylcholine↗

Localization of epileptic foci in children.

Electroencephalographic and neuro-imaging data were correlated with the lobe of cortical resective surgery among 48 patients age 16 years or less with medically intractable partial seizures. Decisions about surgery were determined by clinical judgement and therefore our results reflect the weight of these factors in clinical practise. Neuro-imaging disclosed a lesion over the operated lobe in 32 patients (67%). Multiple EEGs demonstrated a most active spike focus over the operated lobe in 32 patients (67%). Although only 34 patients had a recorded seizure, this clearly arose over the operated lobe in 23 (68% of the 34 patients; 48% of series). Persistent focal delta activity appeared in 20 of the 48 patients (42%). No instance was encountered in which false lateralization of regional or focal interictal EEG spikes or delta when normal apiculate phenomena were excluded. Thus, the scalp EEG remains a valuable guide to epileptogenesis, even among young patients, and should continue to play a major role in determining the region of cortical resection. Effectiveness of cortical resection for seizure control was clearly better among patients with a single major lesion and among those with normal intelligence.

Cerebral Cortex↗

Regulation of spontaneous opening of muscarinic K+ channels in rabbit atrium.

1. Intracellular mechanism(s) for controlling the opening of muscarinic K+ channels in the absence of an applied muscarinic agonist were studied in rabbit atrium by applying the patch clamp technique to isolated single myocytes. 2. In the cell-attached patch configuration, currents due to the activity of both the muscarinic K+ channel and the inward rectifying K+ channel were recorded. However, while the inward rectifying K+ channel currents were observed in only ten patches of 211 examined, spontaneous opening (i.e. in the absence of a muscarinic agonist) of the muscarinic K+ channel currents was observed in all patches examined in these atrial cells. 3. The single-channel currents due to spontaneous opening of muscarinic K+ channels were identified on the basis of their very similar conductance and gating properties to the unitary events which have been recorded when 0.5 microM-acetylcholine is included in the pipette and 10 microM-GTP is present in the internal side of the patch membrane. 4. Although the spontaneous opening of the muscarinic K+ channels disappeared soon after excision of the patch membrane, this type of channel activity reappeared following application of ATP and MgCl2 to the internal side of the torn-off patch, as expected from previous publications. 5. The K+ channel activity induced by the ATP and Mg2+ (measured as the product of the number of channels, N, times the probability of opening, Po) was strongly dependent upon concentration of free Mg2+; it was half-maximal at 2.2 x 10(-4) M [Mg2+]i. However, after the muscarinic K+ channels had been activated by 100 microM-guanosine 5'-O-3-thiotriphosphate (GTP gamma S) together with ATP and Mg2+, an increase in the Mg2+ concentration from 5.5 x 10(-5) to 2 x 10(-3) M failed to enhance this channel activity. 6. Pertussis toxin, which is known to uncouple muscarinic receptors from associated G-proteins (G(i) or G(o)), failed to inhibit the ATP- and Mg(2+)-induced activation of this K+ channel in the absence agonists. 7. In experiments made to test whether the Mg(2+)-ATP requirement results from an obligatory phosphorylation reaction, ATP was replaced with adenylyl-imidodiphosphate (AMP-PNP), an analogue of ATP which is resistant to hydrolysis. This K+ channel activity was not present when ATP was replaced with AMP-PNP.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗

Rheological measurement of blood coagulation in vascular vessel model tube consisting of endothelial cells monolayer.

An in vitro experimental system was developed to study the interaction between endothelial cells and blood as an early event in coagulation. A designed vascular vessel model tube is composed of a monolayer of bovine aorta endothelial cells (BAECs) cultured on an inner surface of a glass tube by means of a rotatory cultivation method. The change of fluidity during coagulation of blood in the tube was measured by a rheological technique. The rate of coagulation of blood in contact with endothelial cells was affected by cell culture conditions such as cell age, passage number of BAECs and substrate beneath endothelial cells. Fibrinolytic activity of the cells was examined by the rheological method. The present experimental system would be useful in examining the mechanism of blood coagulation based on the interaction between blood and endothelial cells as well as in evaluating endothelial cell functions.

Animals↗

Procoagulant activity of collagen. Effect of difference in type and structure of collagen.

Procoagulant activities of different types and structures of collagen were examined. Collagens used were types I (including its methylated and succinylated forms), II, III, IV and V. Each collagen was coated on an inner surface of a glass tube. The change of fluidity during coagulation of blood in the tube was measured by means of a new rheological technique. For monomeric collagen, the procoagulant activity of the succinylated form (negatively charged) was higher than that of the methylated form (positively charged). The procoagulant activity of type IV (dry) was lower than that of other types of collagen. For fibrillar collagens, the initiation of coagulation for type V (non-banded) was fairly delayed compared to those for types I, II and III (banded). An increase in water content in both monomeric and fibrillar forms promoted procoagulant activity. For most of the collagen forms, the addition of factor XII inhibitor (Polybrene) to blood brought about a remarkable delay of the initiation of coagulation, suggesting that the activation of factor XII on the collagen surface is one of main factors governing procoagulant activity. In addition, our data suggest that large numbers of adherent platelets to the collagen surface promote activation of the intrinsic coagulation system.

Adult↗

An improved method for isolating cardiac myocytes useful for patch-clamp studies.

An improved method for isolating cardiac myocytes is described. This method consists of the initial dispersion of the myocytes with a new type of collagenase and the following treatment of the cells with a non-specific protease. This method gives more than 80% rod-shaped, viable, and quiescent cells, which are suitable for biochemical as well as electrophysiological experiments. Formation of a G omega seal and subsequent whole-cell recording is easily performed using these cells.

Animals↗

[Hemorheological studies on platelet counts and size in normal pregnancy and pregnancies with preeclampsia and intrauterine growth retardation].

It has been reported that preeclampsia and pregnancy resulting in intrauterine growth retardation (IUGR) are associated with high hematocrits. The relations between hematocrits (Ht) and platelet volumes in normal and abnormal pregnancies were investigated to clarify a hemorheological effect on formation of microthrombus. 1) In normal pregnancy, Ht was decreased from 12-19 weeks gestation and reached its lowest level at 28-31 weeks gestation. The mean platelet volume (MPV) was decreased from 20 to 31 weeks gestation but markedly increased from 38 to 41 weeks gestation. The platelet count (Pl) remained unchanged during pregnancy. 2) In severe type of preeclampsia, at 28-37 weeks gestation Ht and MPV were markedly increased and Pl was markedly decreased at 38-41 weeks gestation as compared with normal pregnancies. 3) Mothers who delivered IUGR had a much higher level of Ht at 28-35 weeks gestation and MPV level from 38 weeks gestation than in normal mothers. As the volume of young platelets is large, increased MPV is suggestive of the occurrence of platelet consumption. From these results, it was suggested that microcirculatory disturbances such as higher blood viscosity due to hemoconcentration and microthrombus formation were related to the onset of preeclampsia or IUGR.

Blood Platelets↗

Tissue extract recovers cardiac calcium channels from 'run-down'.

Effects of cardiac-tissue extract on the activity of L-type Ca2+ channels were investigated in guinea-pig ventricular myocytes with the patch-clamp method. In most patches, Ca2+-channel current recorded with a pipette solution containing 50 mM Ba2+ and 3 microM Bay K 8644 ran down within 5 min after excision of the patches into a solution containing EGTA. This run-down of Ca2+ channels was prevented when patches were excised into a solution containing a supernatant fraction of homogenate of guinea-pig or bovine heart. Furthermore, this tissue extract was able to restore channel activity after run-down. This channel-activating effect of the extract was abolished by heat treatment or trypsin digestion. Fractionation of the extract by gel filtration suggested that the channel-activating factor(s) had an apparent molecular weight of 2-3 x 10(5). These results suggest that some cytoplasmic protein(s) maintains the activity of the cardiac L-type Ca2+ channel.

Adenosine Triphosphate↗

The postictal electroencephalogram.

Postictal phenomena, their laterality and duration, were studied among 51 patients with scalp EEG-recorded seizures whose origin, evolution and postictal phase were clearly depicted. Regional delta was the most common postictal change (PIC), appearing in 29 patients (57%). Regional attenuation occurred in 15 cases (29%) while the EEG reverted immediately to its interictal nature in 16 instances (31%). 'Activation' of spikes postictally occurred after 13 of the 51 seizures (25%), always over the lobe of seizure onset. PIC, whatever its nature, appeared principally or exclusively ipsilateral to seizure origin in all 35 patients. Moreover, when such changes were bilaterally distributed, they persisted longer on the side of seizure origin in all but one case when durations were bilaterally equal. A multiple PIC (greater than 1 phenomenon) occurred after 21 seizures (41%) while a single change appeared after 14 attacks (27%). Multiple changes occurred more commonly when the seizures had evolved to independent bilateral seizures or to grand mal (77%) than when they were confined to the hemisphere of origin (14%). Thus multiple changes were more often distributed bilaterally (90%) than were single changes (29%). Conversely, seizures without any propagation (focal only) were those most likely to show no postictal change. Multiple changes lasted longer (mean 406 sec) than single changes (79 sec), but duration of PIC was unaffected by seizure propagation or its nature, except for grand mal. Seizure duration had no effect on PIC duration except for grand mal attacks whose postictal effects were predictably longer.(ABSTRACT TRUNCATED AT 250 WORDS)

Brain↗

Inhibition of the calcium channel by intracellular protons in single ventricular myocytes of the guinea-pig.

1. The inhibitory effects of intracellular protons (Hi+) on the L-type Ca2+ channel activity were investigated in single ventricular myocytes of guinea-pigs by using the patch-clamp method in the open-cell-attached patch configuration, where 'run down' of the channel was partially prevented. 2. Hi+ reduced the unitary Ba2+ current of the Ca2+ channel by 10-20% without changing the maximum slope conductance. 3. Hi+ did not alter the number of channels in patches containing one or two channels. 4. Hi+ markedly reduced the mean current normalized by the unitary current, which gave the open-state probability multiplied by the number of channels in the patch. The dose-response curve between Hi+ and the open-state probability indicated half-maximum inhibition at pHi 6.6 and an apparent Hill coefficient of 1. 5. Hi+ shifted both the steady-state activation and inactivation curves in a negative direction by 10-15 mV, and the effects were reversible. 6. Hi+ did not affect the fast open-closed kinetics represented by the C-C-O scheme, apart from increasing the slow time constant of the closed time. 7. Hi+ increased the percentage of blank sweeps and reduced that of non-blank sweeps resulting in a decreased probability of channel opening. 8. Photo-oxidation with Rose Bengal abolished the reducing effect of Hi+ on the open-state probability (Po) in two out of ten experiments, suggesting the possible involvement of histidine residues in the Hi+ effect. 9. The above results indicate that Hi+ inhibits the Ba2+ current mainly by affecting the slow gating mechanism of the channel.

Action Potentials↗

Viscoelasticity of fibrinogen solution and of blood during coagulation studied by a new damped oscillation rheometer.

The behavior of a newly developed damped oscillation type rheometer was analyzed for fibrinogen solution and blood during coagulation. This rheometer consists of a cylindrical tube suspended from a torsion wire, that is filled with liquid to be tested. The logarithmic damping factor (LDF) during coagulation for blood and fibrinogen solution was obtained by this rheometer, which was closely related to the changes of viscosity and/or viscoelasticity of the blood sample. The slight increase of LDF prior to the rapid decrease was observed for blood. The increase of LDF would be reflected in the formation of the aggregation structure of red blood cells (rouleaux network) prior to the formation of fibrin network. The value of LDF for fibrinogen solution sharply increased and then decreased through a maximum value with the progress of coagulation, although the change of LDF was remarkably dependent on the fibrinogen concentration. The initial increase in LDF for fibrinogen solution was considered to be due to the formation of small clots in the solution. The decrease in LDF after attaining a maximum value is ascribed to the formation of fully developed fibrin network. The maximum value of LDF during coagulation for fibrinogen solution is higher than that for blood. The behavior was compared with that for non-biological fluids such as viscosity standard liquids and polyvinyl alcohol solution. From those data, it was concluded that the higher value of LDF than that for Newtonian liquids was due to the formation of aggregation structure or inhomogeneous fine clots in the liquid, which was accompanied with the appearance of the elasticity.

Blood Coagulation↗