Tumor-associated mucin-type glycoprotein (CA54/61) defined by two monoclonal antibodies (MA54 and MA61) in ovarian cancers.

Two monoclonal antibodies, MA54 and MA61, were established by immunizing with culture medium supernatants of a lung adenocarcinoma cell line, and a double-determinant sandwich enzyme immunoassay system was developed by using these two monoclonal antibodies. The antigen recognized by this assay (CA54/61) was found to be often high in the sera of several cancers. The antigen recognized by MA54 (CA54) or MA61 (CA61) proved to be carbohydrate chain on a high molecular weight mucin-type glycoprotein, and CA54 has NeuAc alpha 2-6galactose in the terminal residue. CA54/61 was frequently found in the sera of ovarian cancer patients, the positive rate being 67, 64, 40, and 78% in serous, mucinous, endometrioid, and mesonephroid cancers, respectively, when the cut off value was set at mean + 4 SD. Since the positive rate of CA125, which is now the most widely used for the diagnosis of ovarian cancers, is rather low (approximately less than 50%) in mucinous cystoadenocarcinoma, CA54/61 will be of clinical value. In addition, CA61 was detected immunohistochemically in the fetal red blood cells with nuclei, indicating its oncodevelopmental character in nature.

Cancer-associated galactosyltransferase as a new tumor marker for ovarian clear cell carcinoma.

Serum cancer-associated galactosyltransferase antigen (caGT) was assayed in gynecological cancer patients by means of a GT-II-reactive monoclonal antibody (MAb 3872)-based immunoassay. Thirty-six of 47 (75%) ovarian cancer patients showed a significant elevation of caGT in serum above the cutoff level of 200 milliunits/ml (mean +/- 2 SD) determined from normal controls. Particularly, serum caGT levels in eight of nine patients with ovarian clear cell carcinoma were above the cutoff value, and six of them gave more than 200 milliunits/ml. Elevation of caGT in serum from pregnant women was also detected, and the level increased during the course of gestation. Immunohistochemical study revealed that not only various ovarian carcinoma cells in vivo and in vitro, but also syncytiotrophoblast of early gestational placenta, fetal tissues such as mucus-producing cells in the lower alimentary tract, and renal tubules at the 11th week of gestation were stained with MAb 3872, thus indicating its oncofetal character. Compared with CA-125, caGT showed a lower false-positive rate (10%) in benign gynecological diseases, and there was no correlation between caGT and CA-125 values. Therefore, caGT will be a useful tumor marker for ovarian cancers, especially for clear cell carcinoma.

CA54/61 as a marker for epithelial ovarian cancer.

Using a new one-step, double-determinant enzyme immunoassay, we performed quantitative measurements of a mucin-type glycoprotein antigen (CA54/61) that we recently detected in sera of ovarian carcinoma patients. When the cutoff value was set at 12 units/ml, at which a high diagnostic efficiency was demonstrated [or at 20 units/ml (mean + 3 SD of healthy females)], the positive rates of ovarian serous, mucinous, clear cell, and endometrioid carcinomas were 76% (or 63%), 63% (or 55%), 57% (or 52%), and 50% (or 38%), respectively. Even in mucinous cystadenocarcinoma, more than one-half of the cases were positive, indicating the potential utility of the assay in the diagnosis of mucinous tumors. In sera from patients with benign ovarian tumors, only 9% (or 4%) of the cases were positive, indicating the quite high specificity of this test for ovarian carcinomas. To make a comparison between CA54/61 and CA125, we set the cutoff level of CA125 at 110 units/ml, at which value a high diagnostic efficiency was demonstrated [or at 35 units/ml (mean + 3 SD of healthy females)]. When both CA54/61 and CA125 were assessed in sera from 36 patients with mucinous cystadenocarcinoma, the positive rates of CA54/61 and CA125 were 64% (or 56%) and 36% (or 56%), respectively, suggesting that CA54/61 is of clinical value as a new tumor marker for ovarian cancers, including mucinous tumors.

Glycogen metabolism in rat liver during transition from the fed to fasted states.

Hepatic glycogen metabolism was studied in rats during the period of transition from the fed to fasted states. Glycogenic activity was measured in vivo based on the incorporation of [14C]glucose into liver glycogen. Its changes were almost parallel to the changes in glycogen synthase activity. Progressive accumulation of liver glycogen that occurred in the fed state was associated with a proportional increase in glycogenic activity. Within 4 h after the cessation of food intake, glycogenic activity showed a precipitous fall from the peak to its nadir without significant changes in glycogen content. Meanwhile, the glucose concentration in the portal vein decreased. Upon further development of fasting, glycogenic activity displayed a progressive regain, reciprocally as glycogen contents gradually decreased. The precipitous fall of glycogenic activity during the transition from the fed to fasted states was associated with a transient increase in plasma glucagon, and was partly overcome by the injection of anti-glucagon serum. It is concluded that the fall of portal venous concentration of glucose and secretion of glucagon act as a signal to initiate liver glycogen metabolism characteristic of the fasted or postabsorptive state.

Dual mechanisms involved in development of diverse biological activities of islet-activating protein, pertussis toxin, as revealed by chemical modification of lysine residues in the toxin molecule.

Islet-activating protein (IAP), pertussis toxin, is an oligomeric protein composed of an A-protomer and a B-oligomer. There seem to be at least two molecular mechanisms by which IAP exerts its various effects in vivo and in vitro. On the one hand, some of the effects were not significantly affected by acetamidination of the epsilon-amino groups of the lysine residues in the molecule. These include the activities in vitro (1) catalyzing ADP-ribosylation of one of the membrane proteins directly, (2) enhancing membrane adenylate cyclase activity in C6 cells, (3) reversing receptor-mediated inhibition of insulin or glycerol release from pancreatic islets or adipocytes, respectively, and the activities in vivo (4) inhibiting epinephrine-induced hyperglycemia, (5) potentiating glucose-induced hyperinsulinemia, (6) reducing hypertension and increasing the heart rate in genetically hypertensive rats. These activities are concluded to develop as a result of ADP-ribosylation catalyzed by the A-protomer which is rendered accessible to its intramembrane substrate thanks to the associated B-oligomer moiety. Thus, neither the enzymic activity of the A-protomer nor the transporting activity of the B-oligomer needs free amino groups of the lysine residues in the IAP molecule. On the other hand, additional effects of IAP, such as (1) mitogenic, (2) lymphocytosis-promoting, (3) histamine-sensitizing, (4) adjuvant and (5) vascular permeability increasing, were markedly suppressed by acetamidination of the intrapeptide lysine residues. The free epsilon-amino group of lysine would play an indispensable role in the firm (or divalent) attachment of the B-oligomer of IAP to the cell surface that is responsible for development of these activities.

The efficacy of kinesthetic reading treatment for pure alexia.

This paper presents an effective treatment for pure alexia by a type of single-case design, which we termed a "material-control single-case design" [Sugishita et al., Neuropsychologia, Vol. 31, 559-569, 1993]. Two patients with pure alexia were treated using kinesthetic reading (reading by tracing or copying the outline of each letter with the patient's finger). The results clearly demonstrated that both patients significantly improved their reading and copying performances. Their recovery of reading performance arose from improvement in copying. The results of tachistoscopic reading tests suggested that the patient obtained the ability to read without kinesthetic movements.

High dose rate brachytherapy for carcinoma of the uterine cervix: comparison of two different fractionation regimens.

PURPOSE: There is no consensus as to the best dose-fractionation regimen in high dose rate (HDR) brachytherapy for cervix cancer. Since 1983, two fractionation regimens have been used in different time periods at National Cancer Center Hospital, and their treatment results have been compared in terms of 5-year survival, local control, and complication rate to find the better therapeutic regimen. METHODS AND MATERIALS: From November 1983 to October 1990, 130 patients with uterine cervix carcinoma were treated with HDR intracavitary brachytherapy using a remote afterloading system. There were 21 Stage Ib patients, 5 Stage IIa, 29 Stage IIb, 2 Stage IIIa, 68 Stage IIIb, and 5 Stage IVa. The median age was 64 years. The median follow-up time was 50 months. Radiotherapy consisted of external beam irradiation to the pelvis (mean dose of 50 Gy), combined with HDR brachytherapy (mean dose of 20 Gy to point A) given 5 Gy per session twice weekly (group A: 54 patients) or 6 Gy once weekly (group B: 76 patients). RESULTS: The overall 5-year survival was 52% in group A and 72% in group B. Local recurrence rate was 11%, and distant failure rate was 21%, with no difference between the two groups. The complication rate was significantly lower in group B (37%) than in group A (55%). Multivariate analysis has shown that factors affecting survival were stage, brachytherapy dose, and local control status. No factor was predictive of local control, but the external beam radiation dose significantly influenced the risk of complications. CONCLUSION: The once-weekly HDR intracavitary applications combined with properly adjusted external beam pelvic irradiation is a safe and effective treatment for patients with uterine cervix cancer.

Thallium-201 stress scintigraphy in Takayasu arteritis.

Thirty-eight women with Takayasu arteritis were studied using thallium-201 stress myocardial scintigraphy to assess the prevalence and pathophysiology of the perfusion abnormality. Twenty (53%) had abnormal scintigraphic findings (group A). Abnormal scans were divided into 3 groups: permanent defects in 6, reversible defects in 7 and slow washout in 7. The remaining 18 patients had normal scintigrams (group N). Group A had a tendency to be older and to have a high prevalence of complicated significant aortic regurgitation. Interventricular thickness plus left ventricular posterior wall thickness (26 +/- 7 vs 17 +/- 2 mm, p less than 0.01) and left ventricular mass (267 +/- 121 vs 133 +/- 39 g, p less than 0.01) were all greater in group A on echocardiography. The mean value of the central aortic pressure in systole was 170 +/- 15 mm Hg in the 7 catheterized patients in group A. Coronary ostial stenoses were present in 2 group A patients who showed reversible defects on scintigrams. These data indicate that the abnormal perfusion detected by imaging in patients with Takayasu arteritis was responsible for a decrease in coronary reserve or myocardial damage, or both, due to long-standing systemic hypertension or aortic regurgitation. Coronary artery disease should be considered if a reversible defect is present.

Enhanced flow velocity increase through the left ventricular inflow tract of patients with isolated aortic regurgitation.

Twenty-five patients with chronic aortic regurgitation (AR), and 12 control subjects were studied using Doppler echocardiography to investigate the effects of AR on transmitral flow. Peak early filling velocities at the levels of the mitral valve tips (E1) and annulus (E2) were measured, and the transmitral flow restriction index (delta E = (E1-E2)/E2) was obtained. Patients with AR were classified into 2 groups according to the ratio of the cross-sectional area of the regurgitant jet to that of the left ventricular outflow tract. Group I had the ratio less than 0.20, and group II had greater than or equal to 0.20. E2 in group II was lower than in control subjects, whereas E1 was not significantly different in any groups. delta E in group II was higher than in group I or in control subjects (p less than 0.05 and 0.01, respectively). delta E showed a significant correlation with the cross-sectional area ratio in all patients with AR (r = 0.70, p less than 0.01) and in group II (r = 0.82; p less than 0.01). Our data suggest that AR restricts early transmitral filling, and that delta E may indicate the increased driving pressure caused by flow restriction and is a useful hemodynamic index of AR.

The control of the contraction of myocytes from guinea-pig heart by the resting membrane potential.

1. The influence of different holding potentials (-120 to -70 mV) on the contraction of enzymatically dispersed myocytes from guinea-pig hearts was evaluated. Contractions were elicited by repetitive depolarizations to 0 mV at 0.5 Hz. 2. While ineffective at 140 and 5 mmol l-1 [Na+]o and pipette Na+, respectively, depolarization of the resting membrane with the holding potential increased myocyte shortening at reduced Na+ gradients ([Na+]o 70 or [Na+]i 10-15 mmol l-1). Elevated intracellular Na+ after Na(+)-pump inhibition with ouabain 1-10 mumol l-1 was similarly effective with regard to the inotropic response to different holding potentials. 3. At -70 mV holding potential, reduction of [Na+]o from 140 to 70 mmol l-1 increased myocyte shortening and induced an inwardly directed component of the holding current which peaked at -44 +/- 10 pA and declined thereafter in parallel with the inotropic effect. The relation of this inward current to [Ca2+]i was confirmed by experiments at high Ca2+ buffer capacity where [Na+]o reduction induced a Ni(2+)-insensitive, outwardly directed component (36 +/- 15 pA) of the holding current. The observed inward current is suggested to reflect the extrusion of [Ca2+]i in exchange for [Na+]o as a counter-regulatory mechanism which limits the increase of [Ca2+]i. 4. The interventions which increased the strength of the contraction also enhanced the transient tail current after repolarization, suggesting its close relation to [Ca2+]i. This finding confirmed the pattern found with cell shortening. 5. It is concluded that under certain conditions, voltage-dependent and Na(+)-dependent Na(+)-Ca2+ exchange during the interval between the contractions is relevant to the diastolic concentration of [Ca2+]i which in turn determines the accumulation of Ca2+ in the sarcoplasmic reticulum and the magnitude of the subsequent contraction.

Formation of biologically active protein from the subunits of islets-activating protein (IAP), a new protein isolated from Bordetella pertussis.

Based on the finding reported in the preceding paper (Kanbayashi, et al.: J. Biochem) that subunits of islets-activating protein (IAP), a new protein purified from the culture media of Bordetella pertussis, were inactive as such, but regained the original biological activities when recombined, the conditions required for recovery of the biological activities were studied. Essentially the same biological activities as the native IAP were recovered when the smallest subunit, F-3, was incubated with one of the other subunits, F-1 and F-2, at a pH of around 7, at temperatures below 30 degrees C and for longer than 12 h. During the incubation, association products were formed which were isolated by gel filtration as homogenous proteins that consisted of two subunits probably in a molar ratio of 1 : 1. The native IAP (consisting of two IAP subunits including F-3) were equipotent in enhancing insulin secretory responses, in inhibiting epinephrine-induced hyperglycemia, in inducing leukocytosis and in increasing histamine sensitivity in experimental animals.

Biological properties of islets-activating protein (IAP) purified from the culture medium of Bordetella pertussis.

The biological activities were studied of a new protein, islets-activating protein (IAP), purified from the culture medium of Bordetella pertussis. Rats injected intravenously with 1 microgram of purified IAP exhibited markedly enhanced insulin secretory responses to glucose, glucagon, epinephrine, and sulfonylureas over a period from 3 to 10 days after the injection. The degree and duration of the enhancement were proportional to the dose of IAP; the maximal effect induced by 1-2 microgram of IAP persisted for as long as 2 months. There was a highly significant correlation between the enhancement of insulin secretion and suppression of epinephrine hyperglycemia over a wide range of doses of IAP, indicating that suppression of epinephrine hyperglycemia resulted from hypoglycemic action of insulin secreted in response to epinephrine challenge. Additional actions of IAP were observed in mice; mice treated with higher doses of IAP showed symptoms were observed when lower doses of IAP were injected into mice. Thus, it is concluded that IAP is a protein primarily possessing a unique action to potentiate insulin secretory responses of experimental animals to nutritional and hormonal stimuli.

Subunit structure of islets-activating protein (IAP), a new protein isolated from the culture media of Bordetella pertussis.

The subunit structure was studied of islets-activating protein (IAP), a new protein recently isolated from the culture media of Bordetella pertussis and possessing a unique action, i.e., potentiating insulin secretory responses of animals, IAP dissociated into three subunits, F-1, F-2, and F-3, when incubated in 8M urea. Three subunits isolated by chromatography on CM-Sepharose and DEAE-Sepharose columns showed different molecular weights (F-1: 44,000, F-2: 20,000, F-3: 11,000) and different isoelectric points, but similar amino acid compositions. The F-1 subunit consisted of two polypeptide chains linked by S-S bonding(s), while the F-2 and F-3 subunits were single-chain peptides. These subunits, none of which was biologically active alone, associated upon incubation for 2 h at 37 degrees C and regained biological activities after association only when the F-3 subunit was present in the association product. Thus, the F-3 subunit was essential, and the F-1 and F-2 subunits were permissive, for the development of IAP activity in animals.

Prostacyclin and variant angina.

Plasma levels of thromboxane B2 (TXB2) and 6-keto PGF1 alpha in the blood samples taken at the coronary sinus and ascending aorta from twenty-one Japanese patients with variant angina and twenty with effort angina were measured by radioimmunoassay, the objective being to search for the contribution of prostanoids in coronary spasm. The data were compared with data on thirteen subjects free from coronary artery diseases. In coronary sinus blood, plasma TXB2 in patients with effort angina exhibited statistically significant high levels, as compared with data in the controls. These with variant angina also had high levels, albeit without a statistically significant difference. Eight patients with variant angina and for whom the coronary angiogram showed more than 50% of narrowing had statistically significant high levels of TXB2, and the other thirteen with variant angina and normal coronaries or less than 50% of narrowing had the same plasma levels of TXB2 as the controls. In contrast to TXB2, the plasma levels of 6-keto PGF1 alpha in both coronary sinus and aortic blood of patients with variant angina were very low, as compared with normal controls. Statistically significant low levels of 6-keto PGF1 alpha were noted in the coronary sinus blood of patients with variant angina with normal coronaries and in the aortic blood of those with variant angina, as compared with data on the normal controls. Neither ergonovine test nor spontaneous attacks in patients with variant angina revealed characteristic changes in levels of TXB2 and 6-keto PGF1 alpha in the coronary sinus. These data suggest that high levels of TXB2 in patients with atherosclerotic coronaries may be one factor leading to spasm, while low levels of PGI2 may be a contributing factor.

Antiplatelet therapy for atherosclerotic disorders.

Recent studies have revealed the important roles of platelets in atherogenesis via vascular injury. Our in vivo and in vitro studies clearly demonstrate that activated platelets directly inflict injury to vascular endothelial cells, which is associated with a decrease in intracellular cyclic AMP levels in vascular tissues. Antiplatelet therapy is clinically important not only for the prevention of thrombotic episodes but also for the prevention of vascular injury and atherosclerosis. A small dose of aspirin (80 mg) induces clinically hypoaggregativeness of platelets with concomitantly decreased levels of thromboxane A2 in plasma. Our clinical study involving more than 3 years of treatment with small doses of aspirin demonstrated favorable therapeutic effects characterized by hypoaggregation of platelets and increased levels of cAMP and 6-keto PGF1 alpha in plasma which will aid in the prevention of atherosclerosis.

Hypoglycemia induced by alpha-adrenergic stimulation during alkalosis.

Hypoglycemia developed during respiratory alkalosis in fasted rats. This hypoglycemia was markedly potentiated by the simultaneous injection of inhibitors of hepatic gluconeogenesis or a beta-adrenergic blocking agent; was not influenced by anti-insulin serum; was attenuated by hexamethonium; and was abolished by an alpha-adrenergic blocking agent. The rate of removal of injected glucose by peripheral tissues increased during alkalosis in insulin-deficient rats. The uptake of [14C]-glucose by the adipose tissue in vivo, which is stimulated by a very minute amount of insulin, was also stimulated during alkalosis whether or not the circulating insulin was neutralized with anti-insulin serum. It was concluded that, in alkalotic rats, blood glucose is rapidly utilized by peripheral tissues dependent on alpha-adrenergic stimulation, but without mediation of insulin and that this leads to development of hypoglycemia.

An involvement of alpha-adrenergic stimulation in exercise-induced hypoglycemia.

Hypoglycemia developed in fasted rats during forced swimming. This hypoglycemia was mostly abolished by phentolamine, an alpha-adrenolytic agent, or by hexamethonium; was potentiated by propranolol, a beta-adrenolytic agent, of by 5-methoxyindole-2-carboxylic acid, a gluconeogenic inhibitor; and was not affected by anti-insulin serum. The turnover rate of blood glucose estimated from the decay curve of blood [14C]glucose increased significantly during exercise. There was a slight but significantly increase during exercise in the transfer of 3-O-methyl-[14C]glucose into muscle and adipose tissues, when it was corrected for by [3H]mannitol transfer to the same tissues. It is concluded that the alpha-receptor-mediated action of endogenous catecholamine stimulates peripheral glucose utilization leading to hypoglycemia during exercise. The action of alpha- and beta-adrenergic mechanisms, directly on peripheral tissues or via insulin secretion, in fine regulation of blood glucose level is discussed.

Protective role of nitric oxide synthase against ischemia-reperfusion injury in guinea pig myocardial mitochondria.

In guinea-pig myocardial mitochondria preparation, lowering the Ca2+ concentration or pH level in the perfusate rapidly elevated the fura-2 Ca2+ signal ([Ca2+]m). Pretreatment with 10(-4) M L-Arg inhibited the rapid [Ca2+]m influx, whereas administration of 10(-4) M L-NAME did not, suggesting some association between nitric oxide (NO*) synthase (NOS) activation and Ca2+ kinetics in mitochondria. Immunoblotting analysis showed that endothelial (e)-NOS was present in mitochondria, but not inducible (i)-NOS or brain (b)-NOS. Electron microscopy observations revealed that the e-NOS antibody-reactive site in the mitochondria was the inner cristae. The production of reactive oxygen species and NO* in isolated mitochondria was detected by the spin trapping technique with electron paramagnetic resonance (EPR) spectrometry. Pretreatment with 10(-5) M S-nitroso-N-acetyl-DL-penicillamine (SNAP) and 10(-5) M 3-[2-Hydroxy-1-(1-methylethyl)-2-nitrosohydrazino]-1-propananin e (NOC 5), which spontaneously generate NO*, completely inhibited the [Ca2+]m uptake. In addition, N-morpholino sydnonimine hydrochloride (SIN-1) (10(-5) M), which simultaneously generates NO* as well as *O2- and peroxynitrite anion (ONOO-), inhibited the increase in [Ca2+]m. ONOO- (3 x 10(-4) M) itself also inhibited this increase. Pretreatment with the *O2(-)-scavenger manganese superoxide dismutase or catalase (200 units/ml) completely inhibited the increase in [Ca2+]m caused by lowering of either the Ca2+ concentration or the pH in the perfusate. These results suggested that the formation of reactive oxygen species promoted the [Ca2+]m influx. The agents that inhibited the [Ca2+]m influx improved contractility even in Langendorff preparations after ischemia. Based on these findings, we concluded that e-NOS exists in mitochondria and that NO* may play an important protective role in reperfusion cardiac injury after ischemia, by inhibiting the Ca2+ influx into mitochondria which are otherwise damaged by *O2-.

The effect of low intensity bicycle exercise on the insulin-induced glucose uptake in obese patients with type 2 diabetes.

OBJECTIVE: The present study was undertaken to reveal the effect of low intensity bicycle exercise on the insulin-induced glucose uptake in obese patients. SUBJECTS AND METHODS: Seven obese men with Type 2 diabetes (OBDM) and seven healthy young men (HY) participated in this study. The glucose infusion rate (GIR) was determined by glucose clamp procedure at an insulin infusion rate of 40 mU m-2 min-1 (plasma insulin concentrations: 700-800 pmol l-1). Confirming stabilized GIR, a 30-min bicycle exercise was performed during the glucose clamp which was continued for 120 min after exercise. RESULTS: Average GIR in OBDM for last 30 min prior to exercise were significantly lower than HY (28.3 +/- 1.7, 47.4 +/- 1.8 mumol kg-1 min-1 respectively, P < 0.05). GIR abruptly increased during exercise and gradually decreased after exercise to the nadir almost at the time from 30 to 60 min in recovery period in both groups. GIR in OBDM, however, gradually increased significantly over pre-exercise levels (P < 0.05), following exercise and reached the same levels compared to HY after 80 min of recovery period. CONCLUSION: These results indicated that in obese Type 2 diabetes, 30 min of low intensity bicycle exercise significantly enhances the lower level of insulin-induced glucose uptake shortly after exercise and might be useful for the treatment of post-prandial hyperglycemia.

Cytoprotective effect of TRK-100, a prostacyclin analogue, against chemical injuries in cultured human vascular endothelial cells.

We studied the cytoprotective effect of TRK-100, a chemically stable analogue of prostacyclin (PGI2), in the cultured human endothelial cells from umbilical vein. TRK-100 (10 and 100 nM) stimulated significantly proliferation of endothelial cells but did not affect PGI2 production in endothelial cells. Exposure of cultured endothelial cells to homocysteine (2.5 mM) or glucose (50 mM) caused concentration-dependent cytotoxicity, as evidenced by a decrease in number of viable cells. When endothelial cells were treated with TRK-100 simultaneously or prior to, but not after, exposure to injury substances, decreases in viable cell were significantly suppressed. The protective effect of TRK-100 against homocysteine-induced cytotoxicity also appeared in endothelial cells treated with acetylsalicylic acid, suggesting that endogenous PGI2 did not involve in the protective effect of TRK-100.