Biomedical subjects

K Sakai

Publications and source records attributed to K Sakai.

At least 955 records · Page 53Linked to original sources

PubMed1989

A critical role of the posterior hypothalamus in the mechanisms of wakefulness determined by microinjection of muscimol in freely moving cats.

In order to determine critical sites within the hypothalamus responsible for the induction and maintenance of wakefulness (W), we performed microinjections of muscimol, a potent gamma-aminobutyric acid (GABA) agonist, in various lateral hypothalamic regions of freely moving cats. We found that bilateral injections of a small amount of muscimol (0.1-1.0 micrograms/0.5 microliters) in the preoptic and anterior hypothalamus and rostral mesencephalic tegmentum resulted in increased vigilance and insomnia. In contrast, microinjections of muscimol in the middle and anterior parts of the posterior hypothalamus induced long-lasting behavioral and electroencephalographic signs of sleep with short latency. The hypersomnia was characterized by a significant increase in both light and deep slow wave sleep (SWS), and a nearly complete suppression of paradoxical sleep (PS). Animals with muscimol microinjections in the ventrolateral part of the posterior hypothalamus, however, exhibited increased SWS followed by a significant increase in PS. When injected into the posterior hypothalamus of insomniac cats pretreated with p-chlorophenylalanine (PCPA), muscimol induced not only SWS but also PS with short latency. The present data thus support the hypotheses that the posterior hypothalamus plays a critical role in the mechanisms of W and that sleep might result from functional blockade of the hypothalamic waking center.

Animals↗

PubMed1989

A possible involvement of oxygen free radicals in the development of myocardial acidosis during coronary occlusion in dogs.

The present study was designed to examine whether free radical scavengers attenuate myocardial acidosis induced by partial occlusion of the coronary artery in dogs. The myocardial pH was determined by a micro glass pH electrode inserted in the endocardial layers of the left ventricular wall perfused by the left anterior descending coronary artery. The left anterior descending coronary artery was occluded for 90 min incompletely so that the flow would be 1/2-1/3 the original flow. The myocardial pH before partial occlusion was 7.54-7.55. Partial occlusion decreased the flow in the left anterior descending coronary artery by 49.3-64.9% and the myocardial pH by 0.71-0.76, and increased the ST segment (surface electrocardiogram) by 6.3-9.3 mV. Saline (0.5 ml/kg), recombinant human superoxide dismutase (70,000 or 210,000 U/kg), or catalase (55,000 or 165,000 U/kg) was injected intravenously 30 min after partial occlusion. The injection of recombinant human superoxide dismutase or catalase alone did not restore the myocardial pH that had been decreased by coronary occlusion. The combined injection of recombinant human superoxide dismutase (70,000 U/kg) + catalase (55,000 U/kg), however, restored the myocardial pH without restoration of ST segment. In conclusion, recombinant human superoxide dismutase + catalase attenuated myocardial acidosis during ischaemia, suggesting a possible involvement of oxygen free radicals in the development of myocardial acidosis (especially in the endocardial layers) during ischaemia.

Acidosis↗

PubMed1989

Unitary characteristics of presumptive cholinergic tegmental neurons during the sleep-waking cycle in freely moving cats.

A total of 260 neurons were recorded in the rostral pontine tegmentum of freely moving cats during the sleep-waking cycle. Of these, 207 neurons (80%) were located in the dorsal pontine tegmentum containing monoaminergic and choline acetyltransferase (ChAT)-immunoreactive, or cholinergic neurons. In addition to presumably monoaminergic PS-off cells (n = 51) showing a cessation of discharge during paradoxical sleep (PS) and presumably cholinergic PGO-on cells (n = 40) exhibiting a burst of discharge just prior to and during ponto-geniculo-occipital (PGO) waves, we observed tonic (n = 108) and phasic (n = 61) neurons exhibiting, respectively, tonic and phasic patterns of discharge during wakefulness and/or paradoxical sleep. Of 87 tonic cells histologically localized in the dorsal pontine tegmentum rich in cholinergic neurons, 46 cells (53%) were identified as giving rise to ascending projections either to the intralaminar thalamic complex (n = 26) or to the ventrolateral posterior hypothalamus (n = 13) or to both (n = 9). Two types of tonic neurons were distinguished: 1) tonic type I neurons (n = 28), showing a tonic pattern and high rates of discharge during both waking and paradoxical sleep as compared with slow wave sleep; and 2) tonic type II neurons (n = 20), exhibiting a tonic pattern of discharge highly specific to the periods of paradoxical sleep. Tonic type I neurons were further divided into two subclasses on the basis of discharge rates during waking: a) rapid (Type I-R; n = 17); and b) slow (Type I-S; n = 11) units with a discharge frequency of more than 12 spikes/s or less than 5 spikes/s, respectively. Like monoaminergic PS-off and cholinergic PGO-on cells, both tonic type II and type I-S cells were characterized by a long spike duration (median: 3.3 and 3.5 ms), as well as by a slow conduction velocity (median = 1.8 and 1.7 m/s). In the light of these data, we discuss the possible cholinergic nature and functional significance of these ascending tonic neurons in the generation of neocortical electroencephalographic desynchronization occurring during waking and paradoxical sleep.

Animals↗

PubMed1989

A noise-free molecular hybridization procedure for measuring RNA in cell lysates.

A solution hybridization technique was designed to measure RNA abundance in crude cell lysates and at the same time to maximize confidence that signals resulted from true molecular hybridization. Cell lysates were prepared in 5 M guanidine thiocyanate, then RNA molecules in the lysates were hybridized with two probes, a 32P-labeled RNA "label probe" which provided signal and an oligodeoxyribonucleotide "capture probe" containing a poly(dA) tail which provided a mechanism for selective purification. Ternary hybrids were "captured" on oligo(dT)-coated superparamagnetic beads through a readily reversible interaction with the poly(dA) of the capture probe. RNA did not bind to dT beads through poly(A) under the capture conditions used. Hybrids were purified through cycles of capture on and release from dT beads, with each cycle yielding a 100- to 1000-fold reduction in noise (unhybridized label probe) and a 50-90% recovery of signal (hybridized label probe). Noise was driven below detectable limits after three cycles of capture, thereby improving the sensitivity of measuring target RNA. As few as 15,000 target molecules, 15 fg of a 3-kb RNA, was detectable in the equivalent of 2 x 10(6) cells in concentrated cell lysates (10(8) cells/ml). Since hybridization with both probes was required in order to yield a signal, hybridization specificity could be adjusted with either or both probes. The greater specificity and lack of noise increased confidence that the signal was proportional to the amount of RNA of interest.

Guanidines↗

PubMed1989

Measurement of human portal blood flow by continuous thermodilution.

We wanted to use the Ganz thermistor catheter, developed to measure blood flow in human coronary sinuses, to measure portal venous flow, which is greater. We prepared a model circuit with a high flow rate and studied the method for suitability and reproducibility of the measurements. Correlation was close between the measured and calculated flow rates when the thermal indicator was injected at the rate of 48 ml/min. Measurements were highly reproducible. The catheter was then tested clinically. It was inserted into the portal trunk during percutaneous transhepatic catheterization to measure the portal venous flow. Measurements in patients with cirrhosis of the liver were close to those reported by other workers, and were reproducible. The results suggest that this method will be useful for the continuous local measurement of portal venous flow that varies with time, and will provide information about portal circulation in hepatic disease.

Adult↗

PubMed1989

Methotrexate-resistant mechanisms in human choriocarcinoma cells.

Choriocarcinoma cells grown in the presence of methotrexate (MTX) developed resistance in two ways. HCCM-derived sublines (relatively high MTX resistance) produced enhanced levels of dihydrofolate reductase (DHFR) and had impaired transport of MTX. Altered transport was the primary determinant of response in CC1-derived sublines (low MTX resistance). Since the selection procedures used were identical, it was assumed that altered MTX transport was insufficient to account entirely for the various degrees of resistance. An increased level of DHFR activity was necessary for the development of high MTX resistance. The overproduction of DHFR was the consequence of amplification of the DHFR gene sequence. The incidence of double minutes (DMs) in metaphase paralleled the degree of resistance. However, DMs were also present in cells not showing DHFR gene amplification. Mechanisms other than DHFR gene multiplication were responsible for the de novo synthesis of DMs.

Blotting, Southern↗

PubMed1989

Simple and specific assessment of Ca-entry-blocking activities of drugs by measurement of Ca reversal.

A new, simple, and specific screening method for Ca-entry blocker is reported. In the presence of various blockers of Ca-entry (Ca-channel blockers), Ca2+ (1 x 10(-4) M) causes Ca reversal, i.e., it causes relaxation of rat uterine smooth muscle that has been tonically contracted with oxytocin in Ca-free medium (Ca-free contraction) after prolonged preincubation with 3 mM EGTA. The potencies of various drugs to cause Ca reversal, assessed on an all-or-none basis, were found to reflect their activities of blocking Ca entry. The potencies of drugs for blocking Ca entry via voltage-operated Ca-channels could be determined in 125.6 mM K+ Ca-free medium. Drugs that inhibited Ca-free contraction directly have some other antispasmodic activity. Conventionally, activity for blocking Ca entry is screened by measuring inhibition of Ca-induced contraction, but true Ca-entry blocking activity cannot be differentiated clearly from other antispasmodic activity when a drug has both activities. However, by measuring Ca reversal, activity for blocking Ca entry only can easily be found, when done in solution with high K+ content for blocking voltage-operated channels and when done in solution with normal K+ content for blocking voltage-independent channels.

Animals↗

PubMed1989

Functional evaluation of sympathetically mediated responses in in vivo lower urinary tract of dogs.

An in vivo procedure for evaluating local effects of alpha-adrenoceptor stimuli on the lower urinary tract was developed in anesthetized dogs. Electrical stimulation of hypogastric nerve at varied frequencies (1, 2, and 4 Hz) and intraarterial (i.a.) administration of an alpha 1-adrenoceptor agonist, phenylephrine (0.3, 1, and 3 micrograms) to the urethra and bladder through the cannulated right external iliac artery caused reproducible frequency- or dose-related increases in intraurethral pressure (IUP). Intrabladder pressure (IBP) was increased by the nerve stimulation but not by i.a. phenylephrine. Acetylcholine (10 micrograms) given i.a. elicited increases in both IUP and IBP. Prazosin (0.1, 1, and 10 micrograms/kg, i.v.) dose-dependently suppressed the urethral contractile responses to the nerve stimulation and i.a. phenylephrine, but it failed to affect the bladder contraction evoked by the nerve stimulation. The results suggest that the urethral contractile responses to hypogastric nerve stimulation as well as i.a. phenylephrine were mediated via alpha 1-adrenoceptors, whereas the IBP increasing effect of hypogastric nerve stimulation was not mediated via alpha 1-adrenoceptors.

Acetylcholine↗

PubMed1989

"Ca reversal": inhibition by submicromolar Ca2+ concentrations of contraction of smooth muscle and its use for assay of Ca channel blockers.

1. Ca ion, which is a trigger of many physiological responses, also acts as an inhibitor of responses at lower concentrations than those that act as triggers in rat uterine smooth muscle. 2. The contraction induced by oxytocin of uterine smooth muscle that had been preincubated in Ca-free solution with 3 mM EGTA for 1 hr was reversed by addition of a resting level of Ca ion (10(-8) - 10(-7) M). 3. This relaxation by Ca ion was named "Ca reversal". 4. This finding suggests that Ca ion at low concentrations has an inhibitory action. 5. Since Ca reversal is manifested in the presence of Ca antagonists, we tested the availability of this phenomenon for assay of Ca antagonistic activity. 6. This system is very simple because the judgement is all or none, and it is very useful because other antispasmodic actions can be determined simultaneously. 7. The ED50 values of various drugs for Ca reversal show their Ca antagonistic activities. 8. Furthermore, the value in high K+ solution shows the selectivity of a Ca antagonist for voltage-operated Ca channels. 9. Thus this system is very useful for assay of Ca antagonistic activities, which are very important for drug therapy in a wide variety of diseases.

Calcium↗

PubMed1989

Isolation of cDNAs encoding a substrate for protein kinase C: nucleotide sequence and chromosomal mapping of the gene for a human 80K protein.

An acidic phosphoprotein of Mr 80,000, the 80K protein, is a substrate for protein kinase C in fibroblasts and epidermal carcinoma cells. We purified the 80K protein from human squamous carcinoma Ca9-22 cells and fractionated it into two distinct molecular species, designated the 80K-L and 80K-H proteins. The amino acid sequences of the NH2-terminal region and cyanogen bromide-cleaved fragments of the 80K-H protein were determined and a corresponding oligonucleotide sequence was synthesized. Using this as a probe, two cDNA clones, lambda 80H-1 and lambda 80H-2, were selected from a lambda gt10 cDNA library from human A431 cells. The nucleotide sequence has an open reading frame of 1581 nucleotides encoding a protein of 527 amino acids. The deduced amino acid sequence revealed an extremely Glu-rich region. RNA blot analysis with the lambda 80H-1 cDNA clone detected two polyadenylated transcripts of 2.3 and 3.5 kb in Ca9-22 cells. Spot blot hybridization using flow-sorted human chromosomes provided evidence that the gene (G19P1) encoding 80K-H protein maps to human chromosome 19.

Amino Acid Sequence↗

PubMed1989

Pharmacological characterization of sodium channels in the primary culture of individual Drosophila embryos: neurons of a mutant deficient in a putative sodium channel gene.

Sodium channels in Drosophila embryonic neurons were characterized pharmacologically in the primary culture of individual gastrulae. In normal cultures, presence of sodium channels was demonstrated by neuronal degeneration in the presence of veratridine and ouabain, which was inhibited by tetrodotoxin. Embryonic neurons of Df(2R)M-c33a homozygotes that lack a putative sodium channel gene at 60E region showed normal neurotoxin sensitivity. Therefore, sodium channel genes other than 60E must be functional at this developmental stage. We also examined para(ts1) and nap(ts) mutants and found that they were also sensitive to the neurotoxins. To determine the genotypes of single embryo cultures by histochemical staining, we utilized a special chromosome bearing a hsp70-lacZ fusion gene. The lacZ expression in the culture was studied in detail.

Animals↗

PubMed1989

Prevention of experimental encephalomyelitis with peptides that block interaction of T cells with major histocompatibility complex proteins.

Two synthetic immunodominant and nonencephalitogenic peptides of myelin basic protein, N1-20 and AcN9-20, effectively compete with an encephalitogenic peptide, AcN1-11, in an in vitro T-cell response restricted by class II major histocompatibility complex products (I-Au). These mutant peptide constructs, which do not occur in nature, also compete with the self-antigen for the in vivo induction of T cells primed with the encephalitogen AcN1-11. By using these nonpathogenic competitor peptides, it is possible to prevent the development of a prototypic T-cell-mediated autoimmune disease, experimental allergic encephalomyelitis. These results suggest possibilities for the utilization of competitor peptides for therapy of T-cell-mediated autoimmune diseases linked to specific major histocompatibility complex genes.

Amino Acid Sequence↗

PubMed1989

Plasma cholecystokinin and pancreatic polypeptide responses after ingestion of a liquid test meal rich in medium-chain fatty acids in patients with chronic pancreatitis.

Plasma cholecystokinin (CCK) and human pancreatic polypeptide (hPP) responses after ingestion of a liquid test meal rich in medium-chain fatty acids (MCFA) were studied in patients with chronic pancreatitis with or without diabetes mellitus (DM). Integrated response of plasma CCK was significantly lower in patients with chronic pancreatitis and DM than in the two other groups. There was no statistically significant difference between the healthy control subjects and the patients with chronic pancreatitis without DM in the integrated responses of hPP and plasma CCK. These results indicate that diabetic patients with a greatly destroyed pancreas do not release as much CCK as do nondiabetic patients with a mildly impaired pancreas. An MCFA meal is therefore considered safe in patients with a mildly impaired pancreas. For diabetic patients, however, care should be taken not to exacerbate the DM.

Adult↗

PubMed1989

Demonstration in Tupaia papillary muscle preparations of alpha-adrenoceptors mediating positive inotropic effects: comparison with guinea-pigs.

1. Positive inotropic responses to alpha- and beta-adrenoceptor agonists of isolated papillary muscles from the tree shrew (Tupaia), were compared with those from guinea-pigs. 2. In Tupaia, the concentration-response curve for phenylephrine, unlike that for isoprenaline, was not affected by pindolol in a concentration (10(-8) M) sufficient to block beta-adrenoceptor-mediated responses, but it was significantly shifted to the right by phentolamine (10(-6) M). In guinea-pig papillary muscles, however, the concentration-response curve for phenylephrine, like that for isoprenaline, was shifted to the right by pindolol (10(-8) M) but was unaltered by phentolamine (10(-6) M). Furthermore, when the mean concentrations of agonists inducing maximal positive inotropic responses were compared (relative to that of isoprenaline = 1.0), phenylephrine was found to be only slightly less potent (0.84 +/- 0.04; n = 5) in Tupaia and much less potent (0.33 +/- 0.06; n = 5) in the guinea-pig. 3. Although in Tupaia papillary muscles the increase in developed tension induced by a combination of phenylephrine and isoprenaline did not significantly differ from that by phenylephrine alone, it was approximately 3 times larger than that produced by phenylephrine alone in guinea-pigs. 4. These results indicate that in papillary muscles from Tupaia, unlike the guinea-pig, the positive inotropic effects of phenylephrine can be mediated by alpha-adrenoceptors.

Adrenergic alpha-Agonists↗

PubMed1989

Attenuation of ischaemia-induced regional myocardial acidosis by bevantolol, a beta 1-adrenoceptor antagonist, in dogs.

In dogs anaesthetized with pentobarbital, the left anterior descending coronary artery (LAD) was occluded for 90 min. so that about 1/2 of the original flow was allowed to flow (partial occlusion). Bevantolol (a beta 1-adrenoceptor antagonist) or propranolol (a reference drug) was injected intravenously 30 min. after partial occlusion. Regional myocardial pH was measured by a micro glass pH electrode inserted in the LAD area. Partial occlusion decreased myocardial pH by 0.62 to 0.74. Bevantolol (1.0 mg/kg) or propranolol (1.0 mg/kg) significantly increased myocardial pH, that had been decreased by partial occlusion, within 60 min. after the injection. Restoration of myocardial [H+] (defined as return towards a lower [H+] to the preocclusion level) (calculated from the pH data) induced by bevantolol and that induced by propranolol were 64.0 and 66.4% (measured 60 min. after the injection), respectively. Bevantolol or propranolol decreased heart rate also. Even in the paced heart, bevantolol restored the myocardial [H+] that had been increased by partial occlusion. These results suggest that bevantolol has a favorable effect on the ischaemic myocardium as has propranolol, and that the pH effect of bevantolol is not primarily due to a decrease in heart rate.

Acidosis↗

PubMed1989

Effectiveness of insulin suppositories in diabetic patients.

Experimental insulin suppositories regulated postprandial hyperglycaemia in diabetic patients. The insulin suppositories seemed also to avoid hyperinsulinaemia. The formulation with more rapid dissolution of insulin, which contained a solid dispersed form of insulin, effectively reduced the insulin dose required.

Adult↗

PubMed1989

Comparative effects of ouabain on isolated papillary muscle from tree shrews, guinea-pigs and rats.

Inotropic effects of ouabain were investigated in the isolated papillary muscle preparations of the tree shrew (Tupaia glis), guinea-pigs and rats. In the guinea-pig and shrew papillary muscles, ouabain at concentrations of 10(-8) to 3 x 10(-7) M caused a concentration-dependent positive inotropic response in a similar magnitude, while in the rat, ouabain at concentrations of 10(-7) to 3 x 10(-6) M elicited negative inotropic one. Either phentolamine or pindolol in a concentration sufficient to block the alpha- and beta-adrenoceptors did not antagonize the positive inotropic effect of ouabain in the papillary muscle preparation of the shrews.

Animals↗

PubMed1989

Contribution of the tissue angiotensin converting enzyme to the antihypertensive effect of altiopril calcium (MC-838) in spontaneously hypertensive rats.

The effect of a new orally active angiotensin converting enzyme (ACE) inhibitor, calcium (-)-N-[(S)-3-[(N-cyclohexylcarbonyl-D-alanyl)thio]-2-methylpropionyl+ ++]-L- prolinate (MC-838, altiopril calcium), on systemic blood pressure (SBP) and tissue ACE activity has been examined in conscious spontaneously hypertensive rats (SHRs). MC-838 (3 mg kg-1) given orally to SHRs elicited a long-lasting hypotension lasting over 24 h. With the development of the hypotension, MC-838 significantly reduced ACE activity in the lung, kidney and aorta, but not in the brain and heart. Suppression of plasma ACE and rise of plasma renin activity occurred only transiently at an earlier stage.

Animals↗