The application of freeze-substitution (FS) and freeze-drying (FD) techniques and the protein A-gold-antibody complex immunocytochemical methods are described. The two tissue-preparation techniques produced excellent ultrastructure and topographical fixation of antigens when compared with conventional tissue-preparation techniques. In the FS preparation, however, occasional extragranular immunolabeling was recognized. This may suggest the leakage of antigens from the secretory granules. The FD procedure was considered the best, since such labeling was almost negligible. The protein A-gold-antibody complexes are easily prepared and label the antigens clearly. If the protein A-coated gold particles are saturated with antibodies, there is no interaction between gold particles. Thus, multiple antigens can be determined even in single secretory granules. In fact, we demonstrated intragranular colocalization of immunoreactive oxytocin, labeled with 50-nm gold particles, and immunoreactive methionine-enkephalin, labeled with 15-nm gold particles, in the axonal terminals of the FD-prepared rat neurohypophysis. This study demonstrates the value of the use of gold-antibody complexes for immunocytochemical labeling on FS- or FD-treated tissues.

Adrenocorticotropic Hormone↗

PubMedDate not supplied

Effects of verapamil on myocardial stunning in xanthine-oxidase deficient hearts: pre-treatment vs. post-ischemic treatment.

The role of free radicals and the protective action of calcium antagonists have been established in myocardial stunning in canine hearts, which contain a considerable level of xanthine oxidase, a free radical producing enzyme. However, myocardial stunning in hearts which lack xanthine oxidase and its modification by calcium antagonists in vivo remain uncharacterized. The present study examined this issue using open-chest anesthetized rabbits. Myocardial stunning was induced by a 10-min coronary occlusion and reperfusion. Regional systolic thickening fraction (TF) was determined using an epicardial Doppler sensor, together with other hemodynamic parameters. In untreated control rabbits, recovery of TF from the 10 min transient ischemia was 43 +/- 3% of the baseline at 30 min after reperfusion. Administration of verapamil (200 micrograms/kg bolus plus 40 micrograms/kg/min), which was started before the onset of ischemia and continued until 20 min after reperfusion, significantly improved the recovery of TF to 74 +/- 6% (p < 0.05). A similar improvement in post-ischemic contractile function (TF = 77 +/- 10%) was observed when verapamil was injected at the same rate, but the infusion was discontinued 1 min after the coronary occlusion. Myocardial ATP depletion after the 10 min ischemia was significantly less in the verapamil-pretreated rabbits compared with untreated controls (10.1 +/- 1.0 vs. 6.2 +/- 0.7 mumol/g dry wt., p < 0.05). The difference in TF between the rabbit with and without verapamil treatment could not be explained by afterload reduction. When verapamil (100 micrograms/kg bolus plus 20 micrograms/kg/min) was given during the reperfusion period alone, TF recovery was poorer (TF = 22 +/- 8%) than the control value. Thus, it was concluded that verapamil attenuates myocardial stunning in the hearts with trace levels of xanthine oxidase, and that the beneficial effect is achieved only by pretreatment, not by post-ischemic treatment with verapamil.

Adenosine Triphosphate↗

PubMedDate not supplied

Effect of gonadal steroids on gonadotropin-releasing hormones stimulated human chorionic gonadotropin release by trophoblast cells.

The effect of gonadal steroids on basal and GnRH-stimulated hCG release was studied using collagenase-dispersed trophoblast cells from early pregnancy. Both GnRH and a GnRH superagonist, Buserelin, stimulated hCG release with a similar dose dependency. Progesterone (0.1 to 10 micrograms/ml) inhibited GnRH-stimulated hCG release in a dose dependent manner as well as basal hCG release. Relatively high concentrations of estradiol (10 micrograms/ml) stimulated both basal and GnRH-mediated hCG release and antagonized the inhibitory effect of progesterone on hCG release at 1 micrograms/ml as well as RU486 (1 microgram/ml). These results indicate that progesterone has an important role in both basal and GnRH-mediated hCG regulatory system in the placenta.

Buserelin↗

PubMedDate not supplied

Effects of diacylglycerol and gonadotropin-releasing hormone on human chorionic gonadotropin release by cultured trophoblast cells.

The effect of activation of calcium- and phospholipid-dependent protein kinase (protein kinase C) on human chorionic gonadotropin (hCG) release by cultured trophoblast cells was studied and a role of protein kinase C in the GnRH-mediated hCG release was also evaluated. Both GnRH and 1-oleoyl-2-acetylglycerol (OAG), a protein kinase C activator, stimulated hCG release after 3 h incubation in a dose-dependent manner with ED50 of 55 nmol/l and 4.0 nmol/l, respectively. A tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) also stimulated hCG release while two non-tumor-promoting compounds, phorbol and 4 alpha-phorbol, failed to stimulate hCG release. hCG release by maximal effective dose of GnRH (10 mumol/l) or OAG (1 mumol/l) was further stimulated when cells were incubated with same concentrations of GnRH and OAG. OAG-stimulated hCG release was completely inhibited by a protein kinase C inhibitor, H-7, with ID50 of 23 nmol/l while H-7 did not affect GnRH-mediated hCG release. These results indicate that GnRH-stimulated hCG release is not mediated by protein kinase C pathway, however, the secretion of hCG is also regulated by the mechanism that involves protein kinase C activation.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMedDate not supplied

Detection of bacteria in dialysate and its antibody in long-term hemodialysis patients.

The relationship between endotoxin, which was detected in the dialysate, and incidence of its antibody formation in long-term hemodialysis patients was studied. Bacteria in the dialysate were first obtained, identified, and cultured, and then endotoxin was obtained from the bacteria by extraction with phenol. Using the endotoxin obtained and Escherichia coli J-5 as the antigen, the endotoxin antibody was measured in the serum of long-term hemodialysis patients by enzyme-linked immunosorbant assay (ELISA). It was found that the percentage of endotoxin antibody-positive patients was significantly higher in hemodialysis patients when compared with healthy controls. It was also significantly higher in hemodialysis patients using synthetic polymer membrane dialyzers compared with those using cuprophan membrane dialyzers. These results suggest that endotoxin antibody detected in hemodialysis patients was derived from the dialysate, and that the dialysate must be kept bacteria free, especially in patients using high-flux, highly permeable membrane dialyzers.

Amyloidosis↗

PubMedDate not supplied

Spontaneous and agonist-induced contractions and endothelium-dependent relaxation in aortae from SHRSP and WKY rats under various levels of passive force.

1. The influence of the passive force on the contraction and endothelium-dependent relaxation in aortae of normotensive Wistar Kyoto (WKY) rats and stroke-prone spontaneously hypertensive rats (SHRSP) were compared. 2. Force changes of endothelium-intact and -removed preparations were measured isometrically by a force-displacement transducer. Endothelium-dependent relaxation was observed by applying acetylcholine to the preparation precontracted in the presence of 5 x 10(-7) mol/L noradrenaline. 3. The preparations showed spontaneously developed tension (tone) that increased with the increase in the passive force. The effect of passive force was greater in preparations from SHRSP. Contraction initiated by noradrenaline was also increased by passive force up to 30 mN, then showed a tendency to decrease. 4. Endothelium-dependent relaxation was depressed as the passive force was increased. Preparations from SHRSP showed impaired endothelium-dependent relaxation and were influenced by passive force to a lesser degree when compared with preparations from WKY rats. 5. Relaxation by sodium nitroprusside was influenced by passive force to a much lesser extent than that by acetylcholine. 6. Indomethacin potentiated endothelium-dependent relaxation and blocked the effect of passive force in both preparations. 7. The difference in relaxation and the effect of passive force is primarily caused by the difference in the release of endothelium-derived contracting factor, which is thought to be a product of the cyclo-oxygenase pathway of the arachidonic acid cascade.

Acetylcholine↗

PubMedDate not supplied

A case of Cushing's disease and multiple myeloma.

A 55-year-old female of Cushing's disease associated with multiple myeloma is reported. The association of the two diseases has not been previously noted. Elevated cortisol level was controlled successfully by low doses of adrenocorticolytic agent, o,p'-DDD. However, the exacerbation of multiple myeloma was found to accompany the serum cortisol normalization. Previous reports have shown the good efficacy of combination therapy with melphalan and prednisolone for multiple myeloma. It was suggested that the decrease of endogenous cortisol level might exacerbate the patient's multiple myeloma.

Cushing Syndrome↗

PubMedDate not supplied

[A case of recurrent cerebral hemorrhage considered to be cerebral amyloid angiopathy by cerebrospinal fluid examination].

A 73-year-old man was admitted with gait disturbance and dysarthria. He showed right-side cerebellar ataxia. Computed tomography of brain showed left thalamic bleeding. Nine months later, he was admitted again because of seizure and consciousness disturbance. He had a history of diabetes mellitus and gout for five years, but no hypertension. On physical examination the lungs and heart were normal. On neurological examination, he showed stupor,pupils and eye position were normal. He showed right hemiparesis and urinary incontinence. The deep tendon reflexes were (+) at the upper limbs and (2+) at the right knee and ankle. Blood pressure was 162/88 mmHg and glucose was 275 mg/dl. Other laboratory data were normal. Brain CT showed hemorrhage of the left frontal lobe. The cystatin C level in cerebrospinal fluid was 68 ng/ml. Therefore we suspected cystatin C deposit amyloid angiopathy. In this case, thalamic hemorrhage was initially thought to be amyloid angiopathy. In cases of cerebral hemorrhage in the elderly without hypertension, we must be considered amyloid angiopathy.

Aged↗

PubMedDate not supplied

Immunohistochemical analysis in a case of idiopathic Lennox-Gastaut syndrome.

We herein report a neuropathological and immunohistochemical analysis of a brain from a 25-year-old male with idiopathic type of Lennox-Gastaut syndrome (LGS). The clinical pictures, such as seizure type and progressive mental deterioration with an initial normal psychomotor and mental development in a man were typical of LGS. A routine neuropathological examination showed no pronounced changes, such as neuronal loss, morphologically abnormal neurons, inflammation, vascular changes, Lafora bodies and tumor cells, except that mild gliosis was seen only in CA4 of the hippocampus. Numerous corpora amylacea were observed throughout the cerebral cortices subjacent to the pia mater. An immunohistochemical analysis showed no marked findings for such proteins as glutamate transporters, glutamate decarboxylase, glutamine synthetase, neuronal cytoskeleton proteins and heat-shock proteins. However, intense ubiquitin-immunostained neurons were only found in CA4 of the hippocampus, whereas numerous astrocytes showed a strong immunoreaction for glial fibrillary acidic protein, but showed an exclusively reduced immunoreactivity for metallothionein-I/II, zinc-chelating protein. Our findings thus suggest that the pathology in the hippocampus is either causally or consequentially associated with the seizures occurring in LGS.

Adult↗

PubMedDate not supplied

A comparison of the prognostic significance between the number of metastatic lymph nodes and nodal stage in gastric carcinoma.

BACKGROUND/AIMS: To clarify what staging system of lymph node metastasis is suitable for evaluating prognosis of gastric cancer patients. METHODOLOGY: We analyzed the survival 5 years after operation of 186 advanced gastric cancer patients who underwent potentially curative gastric resection. The following 3 systems were compared using multivariate analyses by the logistic regression model. Nodal status in pathology was classified as follows: (a) nodal stage according to the General Rules for the Gastric Cancer Study of the Japanese Research Society for Gastric Cancer (the Japanese Rules), (b) number of metastatic nodes according to the new UICC staging system (the TNM system), (c) number of metastatic nodes in n1 group of the Japanese Rules (the new classification). RESULTS: The TNM system revealed better results than the nodal stage in sensitivity and -2 log likelihood. The new classification revealed the best result among the 3 systems in sensitivity, specificity, accuracy and -2 log likelihood. CONCLUSIONS: The TNM system is a better prognostic factor than the nodal stage in the Japanese Rules, and the new classification is the best prognostic factor of the above 3 systems in potentially curative advanced gastric cancer patients. Furthermore, the new classification might be useful in comparing data between some facilities.

Adenocarcinoma↗

PubMedDate not supplied

Extraluminal air bubble on computed tomography.

We present two cases of gastrointestinal perforation associated with a tiny extraluminal air bubble on computed tomography (CT). The air bubble was located at the round ligament of the liver in a patient of perforated duodenal ulcer and at the intraperitoneal space adjacent to the perforated site of the anterior gastric wall in the other patient. A small amount of fluid was also seen at the round ligament in the second patient. The region of the round ligament of the liver should be carefully scrutinized for the presence of extraluminal air or fluid in patients with acute abdominal symptomatology.

Adult↗

PubMedDate not supplied

Biochemical studies on phthalic esters. IV. Metabolism of diheptyl phthalate in rats.

Excretion, organ distribution, and metabolism of di(5-methylhexyl) phthalate (DMHP) were studied in male rats. Orally administered 14C-DMHP was rapidly absorbed and 60 and 30%, respectively, of the radioactivity were found excreted into the urine and feces after 7 days. Biliary excretion amounted to about 15% of the dose after 4 days. No specific high concentrations were found in the organs and tissues. Four major metabolites were isolated by HPLC from the ethyl acetate extract of the urine. All of these compounds were monoesters and determined as 5-hydroxy-5-methylhexyl phthalate, 6-hydroxy-5-methylhexyl phthalate, 5-carboxyhexyl phthalate, and 3-carboxypropyl phthalate by spectral analysis (MS, 1H NMR, 13C NMR, UV, IR, etc.). Unchanged DMHP and mono-5-methylhexyl phthalate (MMHP) were found together with a small amount of phthalic acid in the feces. MMHP and its glucuronide conjugate were detected in the bile.

Animals↗

PubMedDate not supplied

Tetraphasic actions of local anesthetics on central nervous system electrical activities in cats.

BACKGROUND AND OBJECTIVES: The effects of seven local anesthetics, mepivacaine, bupivacaine, etidocaine, dibucaine, prilocaine, procaine, and tetracaine, on the central nervous system (CNS) electrical activities were studied in five cats with each agent. METHODS: Brain electrodes were implanted chronically in the cortex, amygdala, hippocampus, and midbrain reticular formation. The cortical, amygdala, and hippocampus electroencephalograms (EEGs), reticular multiunit activity (R-MUA), and heart rate were recorded. Drugs were administered intravenously with constant rates of equipotent doses until EEG seizures appeared. Effects of lidocaine, 1 mg.kg-1.min-1, were studied for comparison. The rates of infusion were: mepivacaine, 1 mg.kg-1.min-1; procaine, 4 mg.kg-1.min-1; bupivacaine and tetracaine, 0.25 mg.kg-1.min-1; etidocaine, 0.5 mg.kg-1.min-1; dibucaine, 0.15 mg.kg-1.min-1; and prilocaine, 3 mg.kg-1.min-1. The effects of 10-15 times higher rates of infusion were also studied in two cats with each drug. RESULTS: During the slow rates of infusion, a tetraphasic sequence of changes, common to all agents, was observed: the initial stage represented by diffuse EEG slowing and a suppression of the R-MUA; the second stage by low voltage fast wave EEG and an activation of the R-MUA; the third stage by reappearance of slow wave EEG and a suppression of the R-MUA; and the fourth stage by an epileptiform EEG and an activation of the R-MUA. These changes in EEG were not typical in some cats administered procaine, bupivacaine, prilocaine, and dibucaine. During the high rates of infusion, suppressive stages in the R-MUA were less dominant and activations dominated. High rates of infusion of mepivacaine produced sudden epileptic activities in EEG. Tetracaine, procaine, etidocaine, and bupivacaine in the high rates of infusion produced slow waves for a short period, prior to seizure activities in the EEG. Dibucaine or prilocaine in high rates of infusion could not produce seizure activities in the EEG, but slow waves and isoelectrical EEG, which were accompanied with idioventricular rhythm followed by ventricular fibrillation. CONCLUSIONS: All local anesthetics have similar tetraphasic CNS actions and whether the expression of CNS intoxication with subconvulsive doses is excitation or suppression may possibly be dependent on the brain drug level and its rate of increase.

Anesthetics, Local↗