Characterization of whole blood aggregation with a new type of aggregometer by a screen filtration pressure method.

A new type of platelet aggregometer of whole blood, based on the screen filtration pressure method, has been developed and characterized. It measures resistance of flow of whole blood samples through a screen of microsieve with 30-microm(2) openings and provides pressure rate as an index of platelet aggregation. On optical microscopic observation, platelet aggregates, but not fibrin fibers, were found to be trapped on microsieves, and the pressure rate and protein amounts on microsieves are correlated. The aggregometer showed good reproducibility for investigations performed on different days. The time course of pressure rates indicated a bell curve change, where the pressure rate was very low immediately after blood collection and gradually increased up to 60 min thereafter. Use of the aggregometer was able to confirm that orally administered aspirin inhibits ADP- and collagen-induced whole blood aggregation as well as platelet aggregation. The results suggest that this platelet aggregometer might be useful in research and clinical diagnosis of thrombotic diseases.

Development and assessment of enzyme immunoassay for platelet-derived microparticles.

Platelet-derived microparticles (PMPs) are released from platelets through the platelet activation by high shear stress, collagen, or calcium ionophore (A23187). PMPs are observed in patients with acute myocardial infarction, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, heparin-induced thrombocytopenia and other thrombotic disorders, but the importance of circulating PMPs in the pathogenesis of these diseases is still debated. Numbers of PMPs are usually determined by flowcytometry (FCM), but easier and reproducible PMP assay systems are needed. To develop a better ELISA for PMPs, we used antibodies against the platelet antigens anti-GPIb (NNKY5-5), anti-GPIIb/IIIa (NNKY2-11, anti-CD41), anti-GPIX (KMP-9), and anti-CD9 (NNKY1-19). PMPs were detected with all combinations of these antibodies, but the ELISA having the highest and most specific absorbance was obtained with a combination of KMP-9 (capture antibody) and NNKY5-5 (detecting antibody). PMPs in blood samples were measured by ELISA and FCM. ELISA correlated with PMPs quantitated by FCM. By shaking ELISA plates during incubation, nonspecific binding of platelets was eliminated. The level of PMPs was not increased in diabetes mellitus, thrombotic thrombocytopenic purpura, antiphospholipid syndrome, or sepsis. The concentration of PMP was elevated in hemolytic uremic syndrome. Activated PMPs were absorbed to 0.8 microm filter, but circulating PMPs were not absorbed. These results suggest that activated PMPs are likely to adhere to leukocytes or endothelial cells at the activation site and that the circulating form of PMPs are likely to be a residue of activated PMPs. To detect only the activated form of PMPs, a new ELISA needs to be developed, and it will likely use a combination of antibodies that detect platelet activation markers such as P-selectin (CD62P) or activated GPIIb/IIIa.

Sperm motion analysis in rats treated with adriamycin and its applicability to male reproductive toxicity studies.

Adriamycin (ADR), an anticancer drug which induces testicular toxicity, was administrated to Slc:SD male rats at doses of 0.5, 1.0, and 2.0 mg/kg intravenously once a week for 4 weeks. The males treated with ADR were mated with untreated females, and sperm analyses (motion, count, and morphology) were performed. Sperm motion was analyzed by Hamilton-Thorne Sperm analyzer (HTM-IVOS) to investigate the useful parameters. Copulated females were necropsied at Day 13 of gestation, and reproductive status was evaluated. In ADR-treated groups, the testicular weight was dose-dependently decreased. Associated with this decrease was a depletion of the number of spermatogonia noted histopathologically at all dosage levels. Sperm morphological abnormalities, which were classified as tailless sperm and/or no-hook head sperm, were increased in both the 1.0 and 2.0 mg/kg groups. The males treated with ADR at 1.0 and 2.0 mg/kg had a decreased number of sperms per cauda epididymis. In sperm motion analysis, decreases in the percentage of motile sperm, percentage of progressive sperm, and sperm velocity (straight line velocity and curvilinear velocity) were noted at 2.0 mg/kg. Impaired fertility was noted at 2.0 mg/kg in the form of decreased numbers of implantations and live embryos, and an increased number of pre-implantation losses. In conclusion, ADR induced deterioration of sperm motion and sperm content, which were responsible for the adverse effect on male fertility. The most sensitive indicators to detect male reproductive toxicity induced by ADR were testicular weight and histopathological findings in the testis. Among the parameters generated by HTM-IVOS, the percentage of motile sperm, the percentage of progressive sperm, and sperm velocity are useful for assessing male fertility.

Spirostanols obtained by cyclization of pseudosaponin derivatives and comparison of anti-platelet agglutination activities of spirostanol glycosides.

Naturally occurring saponins 3 and 4 have a normal type F ring and alpha-arranged CH(3)-21 group. Treatments of pseudosaponin peracetates 18 and 19 derived from 3 and 4, respectively, with alcoholic KOH, followed by acidification with acetic acid, gave spirostanols 20 and 22 having iso type F rings as major products. Structural analyses of sapogenins and saponins derived from pseudo derivatives 11, 12, 18 and 19 were performed by comparisons of their 1H-NMR spectral data and the X-ray analytical data of 3-O-p-bromobenzoyl sarsasapogenin 7, 3-O-acetyl diosgenin 13 and saponin 20. The mechanisms of ring-closure reaction of the side chain at C-22 of pseudosapogenins and pseudosaponins were deduced using stereomodels of the spirostanols derived from 11 under various reaction conditions. Inhibitory activities of saponin diglycosides 3, 4, 20, 21 and 25 on human platelet agglutinations induced by ADP and ristocetin were compared.

An immunohistochemical and ultrastructural study of a follicle-stimulating hormone-secreting gonadotroph adenoma occurring in a 10-year-old girl.

Female gonadotroph adenomas with endocrinological symptoms are uncommon. Six cases of such adenomas have been reported in the literature: two were girls who presented with precocious puberty and four were premenopausal women with accompanying multiple ovarian cysts. We describe here a 10-year-old Japanese girl with a gonadotroph macroadenoma and present detailed morphological findings of the tumor. The patient's chief complaints were nausea, abdominal distention, and abdominal pain. Abdominopelvic ultrasonography and magnetic resonance imaging (MRI) revealed bilateral multiple ovarian cysts. Endocrinological assays showed elevated serum follicle-stimulating hormone (FSH) (33.7 mIU/ml) and estradiol (3840 pg/ml). MRI of the head showed a large pituitary tumor. Two transsphenoidal operations and subsequent radiation therapy were performed. Immunohistochemically, more than half the tumor cells were positive for anti-FSH-beta monoclonal antibody. Ultrastructurally, the tumor cells exhibited a fairly uniform picture of rounded cells. Their nuclei were slightly irregular and contained heterochromatin, and their cytoplasm contained many round, dense core granules, measuring 140-260 nm in diameter, together with well-developed organelles. An in vitro study showed that the tumor cells in primary culture produced FSH (1089.0 mIU/ml). To our knowledge, this is the first immunohistochemical and ultrastructural study of an FSH-secreting gonadotroph adenoma occurring in childhood.

An anti-platelet agent, OPC-29030, inhibits translocation of 12-lipoxygenase and 12-hydroxyeicosatetraenoic acid production in human platelets.

1. In human platelets, arachidonic acid is mainly metabolized by the two enzyme systems; cyclo-oxygenase and 12-lipoxygenase. Cyclo-oxygenase produces prostaglandin H(2) which is further converted to thromboxane B(2). 12-Lipoxygenase synthesizes 12(S)-hydroperoxyeicosatetraenoic acid which is reduced to 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE). 2. An anti-platelet compound, OPC-29030, dose-dependently inhibited 12(S)-HETE production with an IC(50) of 0.06+/-0.01 microM, but not synthesis of thromboxane B(2) in human platelets. Although the compound suppressed 12(S)-HETE production in human platelets, cytosolic 12-lipoxygenase activity was not inhibited up to 10 microM. Essentially identical data were obtained with a 12-lipoxygenase of human erythroleukaemia cells which had megakaryocyte/platelet-like properties. 3. OPC-29030 also suppressed production of 5(S)-HETE, a 5-lipoxygenase product, in rat basophilic leukaemia cells without inhibiting enzyme activity. It has been shown that 5-lipoxygenase binds to membrane 5-lipoxygenase-activating protein (FLAP) to produce 5(S)-HETE, and thus FLAP inhibitor suppresses cellular 5(S)-HETE production. 4. A FLAP inhibitor, L-655,238, suppressed platelet 12(S)-HETE production, but had no effect on the 12-lipoxygenase activity. 5. Western blot analysis showed that platelet 12-lipoxygenase translocated from cytosol to membranes upon thrombin stimulation, and OPC-29030 suppressed this process in a dose-dependent manner. 6. These results suggest that the 12-lipoxygenase of human platelets binds to FLAP or a similar protein, and OPC-29030 suppresses 12(S)-HETE production by inhibiting a certain step of the 12-lipoxygenase translocation.

The recovering effect of betaine on carbon tetrachloride-induced liver injury.

The recovering effect of betaine (derivative of choline) on carbon tetrachloride (CCl4)-injured liver was investigated by oral and intraperitoneal administration of betaine at 24 h after acute CCl4 intoxication. The effect of betaine was estimated by the activity of alanine aminotransferase (ALT) released into the serum, histological score, and the incorporation of S-phase indicator (5-bromo-2-deoxyuridine/5-fluoro-2'-deoxyuridine; BrdU/FdU). A significant decrease in the activity of serum ALT was observed by the intraperitoneal (3 mg/kg body) or oral (15 mg/kg body) administration of betaine. Furthermore, an increase in the uptake of BrdU into the hepatocyte nuclear DNA and a reduction in liver necrosis after the oral treatment of betaine (15 mg/kg body) was observed. From these results, the administration of betaine showed a significant effect in recovering CCl4-injured liver.

Ethanolamine stimulates repair processes in acute CCl4 damage of mouse liver.

Ethanolamine, a positively charged hydrophilic component of the phosphatidylethanolamine (PE) which is also a precursor of phosphatidylcholine (PC), enhances the repair processes 24 h after carbon tetrachloride (CCl4) intoxication of mouse liver. Ethanolamine quickly reduced aminotransferase activity released in the serum, increased the hepatocellular nuclear BrdU uptake, a marker of S phase, and the epidermal growth factor (EGF) receptor content in the liver injured with CCl4. Thus, exogenous administration of ethanolamine may function as a liver proliferating factor in CCl4 intoxicated mouse liver.

The effect of ethanolamine on acute carbon tetrachloride intoxication.

The effects of ethanolamine on injured liver were investigated by oral administration of ethanolamine to male ddY mice 24 h after carbon tetrachloride (CCl4) injection. The serum aminotransferase activities in mice with liver injury were reduced by ethanolamine treatment (10-30 mg/kg body weight). Drastically increased regenerative reaction of ethanolamine treated-CCl4 injured liver was also observed through an increase in 5-bromo-2'-deoxyuridine uptake. ATP concentration in liver tissue was recovered by administration of ethanolamine. These results suggest that oral administration of ethanolamine accelerates recovery from CCl4-induced liver injury.

Serum concentrations of myoglobin vs human heart-type cytoplasmic fatty acid-binding protein in early detection of acute myocardial infarction.

We compared the diagnostic utility of serum concentrations of human heart-type cytoplasmic fatty acid-binding protein (H-FABPc), myoglobin, and their ratio for the early diagnosis of acute myocardial infarction (AMI) in 104 healthy volunteers and 165 patients at admission within 6 h of the onset of chest pain. The ROC curves of the H-FABPc [0.946, 95% confidence interval (CI) = 0.913-0.979] and myoglobin (0.895, 95% CI = 0.846-0.944) between patients with AMI and healthy volunteers were significantly greater than the area under the ratio of myoglobin to H-FABPc (0.823, 95% CI = 0.765-0.881). In 165 patients, the sensitivity (81.8%, 95% CI = 74.2-89.4%), specificity (86.4%, 95% CI = 78.1-94.6%), and predictive accuracy (83.6%, 95% CI = 78.0-89.3%) of H-FABPc > 12 micrograms/L in diagnosing AMI were significantly higher than those of myoglobin, and were similar to those of the combination of H-FABPc > 12 micrograms/L and the ratio < or = 14. We conclude that H-FABPc is a more sensitive and specific marker than myoglobin for the early diagnosis of AMI, and that their ratio cannot give a clear advantage over the measurement of H-FABPc alone.

Early detection of successful coronary reperfusion based on serum concentration of human heart-type cytoplasmic fatty acid-binding protein.

Both human heart-type cytoplasmic fatty acid-binding protein (H-FABPc) and myoglobin are low molecular weight proteins that are abundant in the cytoplasm of myocardial cells. Unlike myoglobin, H-FABPc content in the skeletal muscle is less than in cardiac muscle. To investigate the usefulness of the serum concentration of H-FABPc in the early detection of successful coronary reperfusion, we measured serum concentrations of H-FABPc and myoglobin in 45 patients with acute myocardial infarction treated with intracoronary thrombolysis or direct percutaneous transluminal coronary angioplasty. Coronary angiography was performed every 5 min for reperfusion therapy to identify the onset of reperfusion. Reperfusion, defined as a TIMI grade 2 or 3, was achieved within 60 min of the initiation of reperfusion therapy in 30 patients (the reperfused group), but was not achieved in 15 patients (the non-reperfused group). Blood samples were obtained before initiation of treatment and 15, 30 and 60 min after initiation of treatment in the non-reperfused group. In the reperfused group, samples were obtained before reperfusion and 15, 30 and 60 min after reperfusion. The H-FABPc ratio (the ratio of value after to value before the initiation of treatment or reperfusion) increased sharply after the onset of reperfusion, peaking at 41 +/- 18 min, and decreased rapidly thereafter. The predictive accuracy of an H-FABPc ratio of > 1.8 for the detection of reperfusion within 60 min of the initiation of treatment was 93% at 15 min after reperfusion, 98% at 30 min, and 100% at 60 min. Similar rates of predictive accuracy were observed for a myoglobin ratio > 2.4. The H-FABPc ratio detected successful reperfusion as early as 15 min after the onset of reperfusion and was highly accurate in detecting reperfusion within 60 min of the onset of reperfusion. The predictive accuracy of the H-FABPc ratio was similar to that of the myoglobin ratio for the early detection of successful coronary reperfusion.

Conversion of gamma-glutamylcysteinylethyl ester to glutathione in rat hepatocytes.

The conversion of gamma-glutamylcysteinylethyl ester (gamma-GCE) to glutathione in a reduced form (GSH) was examined using isolated rat hepatocytes pretreated with diethylmaleate, a GSH-depletor. Incubation of hepatocytes with 0.1 and 5.0 mM gamma-GCE (gamma-GCE-hepatocytes) over a 30-min period resulted in time-dependent increases in intracellular GSH and nonprotein-SH (NP-SH) concentrations. Hepatocytes incubated with 5.0 mM but not 0.1 mM GSH over a period of 30 min showed a time-dependent increase in intracellular GSH concentration. In the gamma-GCE-hepatocytes pretreated with bis-(p-nitrophenyl)phosphate (BNPP), a non-specific esterase inhibitor, an enhancement of intracellular GSH concentration was markedly reduced. gamma-GCE concentration in the gamma-GCE-hepatocytes with BNPP pretreatment was significantly higher than that in the cells without BNPP pretreatment, although there was no difference in the total amount of intracellular NP-SH, i.e., gamma-GCE, GSH, gamma-glutamylcysteine, cysteine ethyl ester, and cysteine between both gamma-GCE-hepatocytes. The present results indicate that gamma-GCE is transported into liver cells more easily than GSH itself, resulting in its conversion to GSH via esterase and glutathione synthetase within the cells.

Characterization of cDNA encoding for phosphoglucose isomerase of rice (Oryza sativa L.).

Two types of genes (Pgi-a and Pgi-b) encoding phosphoglucose isomerase (PGI; EC 5.3.1.9) were cloned from cDNA libraries of rice cultured cells (Oryza sativa L.). Pgi-a and Pgi-b consisted of 2132 and 2030 nucleotides, respectively. The homology between these genes was 93.0% at nucleotide level. The homology scores between these genes in protein coding region and 3' non-coding region were 95.6% and 79.4%, respectively. PGI proteins encoded by Pgi-a and Pgi-b consisted of 567 and 568 amino acid, respectively, sharing 95.8% homology at amino acid sequences. Of 11 PGI genes from other plant species and organisms whose amino acid sequences had been determined, a dicotyledonous plant Clarkia lewisii PGI showed the highest homology (about 80%) with rice PGIs. GC contents at the third position of rice PGI genes were about 40%. In order to confirm the enzyme activity of the protein encoded by the rice cDNA, Pgi-a was subcloned into an expression vector, pBluescript II SKp, which was introduced into Escherichia coli. The transformant had an additional PGI activity from Pgi-a.

Stenosis enhances role of platelets in growth of regional thrombus and intimal wall thickening in rat carotid arteries.

The authors present the results of a study in which stenosis was induced, resulting in either thrombus or intimal wall thickening, in rat carotid arteries. At > or = 75% stenosis in mildly denuded arteries, an acute and occlusive thrombus formation was induced, but the thrombus was significantly reduced in thrombocytopenia. Thrombus formation near the site of stenosis decreased with decreasing degree of stenosis, whereas the percent formation in the distal region (percent total thrombus) increased. Numerous mural platelet microthrombi were noted at the distal region of the stenosed arteries. After chronic 50% stenosis of the carotid artery for 2 weeks, significant intimal thickening was observed, without any occlusive thrombus formation. The combination with mild denudation was critical in eliciting the effect of stenosis. The magnitude of intimal growth in the stenosed artery was marked by day 6 and plateaued thereafter, whereas it was slight in nonstenosed arteries. The 5-bromodeoxyuridine index of the cells of the medial layer at day 3 was significantly increased by the stenosis, and the effect was reversed in thrombocytopenia. Complete reendothelialization of the intimal surface was observed by 7 to 10 days after surgery in the stenosed arteries. These findings suggest that the introduction of stenosis in these arteries enhances the interaction of platelets with the damaged arterial walls under abnormal fluid shear and that this enhancement leads to acute and occlusive thrombus formation associated with more marked stenosis as well as to sustained increase of intimal wall thickness in less marked stenosis.

Humoral hypercalcemia of malignancy associated with parathyroid hormone-related protein producing transitional cell carcinoma of the ureter.

A 78-year old male with ureteral carcinoma manifesting hypercalcemia is reported. He was diagnosed as having ureteral carcinoma of the left side 2 years previously and was treated by nephrectomy with ureterovesicostomy. In October 1991, he was admitted for anorexia. A clinical examination revealed recurrence of the ureteral carcinoma with metastasis to the rectum and liver. His serum calcium level was elevated (13.9 mg/dl). In addition to rehydration and furosemide, treatment with eel-calcitonin and prednisolone failed to decrease his serum calcium level. Finally, he was administered mithramycin but he died 13 days later. He had no evidence of bone metastasis or hyperparathyroidism. Nephrogenic cAMP and urinary parathyroid hormone-related protein (PTHrP) were markedly elevated. Immunohistochemical study demonstrated expression of PTHrP in the tumor cells. Thus, the hypercalcemia was thought to be mediated by PTHrP secreted from the neoplastic tumor. Although there have been several reports of ureteral carcinoma associated with humoral hypercalcemia of malignancy, this is considered to be the first case associated with elevation of PTHrP.

[Change in levels of specific terminal sugar residues of glycoproteins in sera of patients with malignant tumor].

Serum sialic acid (N-acetylneuraminic acid, NANA) levels significantly increased in all of patients with stomach cancer, colo-rectal cancer, breast cancer and pancreas cancer. However, the positive rate for serum NANA levels was 35 approximately 60% in these diseases. In contrast, a significant increase in serum NANA-galactose (NANA-Gal) levels was found in only stomach cancer and colo-rectal cancer; especially, the positive rate for serum NANA-Gal levels was 77% in colo-rectal cancer. In colo-rectal cancer, increased serum NANA-Gal levels were decreased after operation. But, in patients with colo-rectal cancer, changes in serum NANA levels following operation were variable. These results indicate that the assay of serum NANA-Gal level provides more important clinical significances for the specificity of cancer and the change of its status than that of serum NANA level.

Assessment of chemoembolization therapy for primary liver cancer using a stabilized adriamycin-lipiodol suspension.

We formulated a new lipiodol-Adriamycin suspension (ADM/lipiodol, 50 mg/10 ml) that remained stable for 48 h (half-life, 25 +/- 3 days). In five cases of hepatocellular carcinoma (HCC) resected after intra-arterial infusion of this agent, the ADM concentration in the tumor was quite high and the tumor necrosis rate was more than 80% on histological examination. Over a 5-year period, 180 patients with unresectable HCC underwent transcatheter arterial embolization therapy (TAE) in the presence or absence of this agent. The regimens consisted of suspension injection alone (A, n = 54), suspension injection + TAE using gelatin sponge (B, n = 29), TAE followed by suspension injection (C, n = 34), and TAE alone (D, n = 63). The estimated 1-year survival values determined for patients treated with these regimens were 70%, 73%, 43%, and 39% respectively, and the corresponding 3-year survival values were 27%, 31%, 15%, and 10%. The survival achieved using suspension injection was thus superior to that obtained using conventional TAE, and combined therapy with suspension injection followed by TAE seemed to enhance survival, although there were some biases in tumor size and in the stage of tumor progression. For patients with tumors measuring 5 cm or more in diameter, the survival obtained using regimen A was lower than that achieved using regimen D, but the combination of TAE and suspension injection improved the 1-year survival value obtained using regimen D from 34% to 52%. For patients with tumors measuring less than 5 cm in diameter, the survival achieved using regimen A was markedly better than that obtained using regimen D, although no difference was found between the survival value achieved using regimen A and that obtained using regimens B and C. On the basis of these results, our newly formulated ADM-lipiodol suspension was surmised to be effective by itself against relatively small HCC tumors, whereas it enhanced the efficacy of conventional TAE in large lesions.

[The inhibitory effect of gamma-glutamylcysteinylethyl ester (gamma-GCE) instillation on experimental diabetic cataract formation in rats].

The effect of the instillation of gamma-glutamylcysteinylethyl ester (gamma-GCE), which has been reported to function as a precursor of glutathione, on cataract formation was examined in rats in which diabetes had been induced by Streptozotocin (STZ). Three days after i.p. treatment with 50 mg/kg body weight of STZ, male Wistar rats aged 6 weeks received instillations of gamma-GCE in solution or liposomes prepared with dipalmitoylphosphatidylcholine (DPPC) for a period of 9 weeks. Cataract formation and development were observed by use of a cataract camera every week. After 9 weeks' observation, the lenses were enucleated and the content of the lens GSH was measured. Instillation of gamma-GCE in solution or liposomes to STZ-diabetic rats not only inhibited cataract formation but also kept lens GSH level almost at the control level. In addition, the inhibitory effect of the instillation of gamma-GCE in liposome was stronger than that of gamma-GCE in solution. The present results indicate that the administration of gamma-GCE in solution or in liposomes inhibits diabetic cataract formation, possibly by preventing lens GSH depletion.