Mutant botrocetin-2 inhibits von Willebrand factor-induced platelet agglutination.

Essentials Botrocetin-2 (Bot2) binds to von Willebrand factor (VWF) and induces platelet agglutination. We identified Bot2 residues that are required for binding to VWF and glycoprotein (GP) Ib. We produced a mutant Bot2 that binds to VWF but inhibits platelet agglutination. Mutant Bot2 could be used as a potential anti-thrombotic reagent to block VWF-GPIb interaction. SUMMARY: Background Botrocetin-2 (Bot2) is a botrocetin-like protein composed of α and ß subunits that have been cloned from the snake Bothrops jararaca. Bot2 binds specifically to von Willebrand factor (VWF), and the complex induces glycoprotein (GP) Ib-dependent platelet agglutination. Objectives To exploit Bot2's VWF-binding capacity in order to attempt to create a mutant Bot2 that binds to VWF but inhibits platelet agglutination. Methods and Results Several point mutations were introduced into Bot2 cDNA, and the recombinant protein (recombinant Bot2 [rBot2]) was purified on an anti-botrocetin column. The mutant rBot2 with either Ala at Asp70 in the ß subunit (Aspß70Ala), or Argß115Ala and Lysß117Ala, showed reduced platelet agglutination-inducing activity. rBot2 with Aspß70Ala showed little binding activity towards immobilized VWF on an ELISA plate, whereas rBot2 with Argß115Ala/Lysß117Ala showed reduced binding activity towards GPIb (glycocalicin) after forming a complex with VWF. rBot2 point-mutated to oppositely charged Glu at both Argß115 and Lysß117 showed normal binding activity towards VWF but no platelet-agglutinating activity. Furthermore, this doubly mutated protein inhibited ristocetin-induced or high shear stress-induced platelet aggregation, and restrained thrombus formation under flow conditions. Conclusions Asp70 in the ß subunit of botrocetin is important for VWF binding, and Arg115 and Lys117 in the ß subunit are essential for interaction with GPIb. Doubly mutated rBot2, with Argß115Glu and Lysß117Glu, repels GPIb and might have potential as an antithrombotic reagent that specifically blocks VWF function. This is the first report on an artificial botrocetin that can inhibit the VWF-GPIb interaction.

Internalization of a novel, huge lectin from Ibacus novemdentatus (slipper lobster) induces apoptosis of mammalian cancer cells.

An N-acetyl sugar-binding lectin (termed iNoL) displaying cytotoxic activity against human cancer cells was isolated from the slipper lobster Ibacus novemdentatus (family Scyllaridae). iNoL recognized monosaccharides containing N-acetyl group, and glycoproteins (e.g., BSM) containing oligosaccharides with N-acetyl sugar. iNoL was composed of five subunits (330, 260, 200, 140, and 30 kDa), which in turn consisted of 70-, 40-, and 30-kDa polypeptides held together by disulfide bonds. Electron microscopic observations and gel permeation chromatography indicated that iNoL was a huge (500-kDa) molecule and had a polygonal structure under physiological conditions. iNoL displayed cytotoxic (apoptotic) effects against human cancer cell lines MCF7 and T47D (breast), HeLa (ovarian), and Caco2 (colonic), through incorporation (internalization) into cells. The lectin was transported into lysosomes via endosomes. Its cytotoxic effect and incorporation into cells were inhibited by the co-presence of N-acetyl-D-mannosamine (ManNAc). Treatment of HeLa cells with iNoL resulted in DNA fragmentation and chromatin condensation, through activation of caspase-9 and -3. In summary, the novel crustacean lectin iNoL is incorporated into mammalian cancer cells through glycoconjugate interaction, and has cytotoxic (apoptotic) effects.

Sialyl-glycoconjugates in cholesterol-rich microdomains of P388 cells are the triggers for apoptosis induced by Rana catesbeiana oocyte ribonuclease.

SBL/RC-RNase was originally isolated from frog (Rana catesbeiana) oocytes and purified as a novel sialic acid-binding lectin (SBL) that displayed strong anti-cancer activity. SBL was later shown to be identical to a ribonuclease (RC-RNase) from oocytes of the same species. The administration of SBL/RC-RNase induced apoptosis (with nuclear condensation and DNA fragmentation) in mouse leukemia P388 cells but did not kill umbilical vein endothelial or fibroblast cells derived from normal tissues. The cytotoxic activity of SBL/RC-RNase was inhibited by desialylation of P388 cells and/or the co-presence of free bovine submaxillary mucin. FACS analysis showed that SBL/RC-RNase was incorporated into cells after attachment to cholesterol-rich microdomains. Addition of the cholesterol remover methyl-ß-cyclodextrin reduced SBL/RC-RNase-induced apoptosis. Apoptosis occurred through the caspase-3 pathway following activation of caspase-8 by SBL/RC-RNase. A heat shock cognate protein (Hsc70) and a heat shock protein (Hsp70) (each 70 kDa) on the cell membrane were shown to bind to SBL/RC-RNase by mass spectrometric and flow cytometric analyses. Quercetin, an inhibitor of Hsc70 and Hsp70, significantly reduced SBL/RC-RNase-induced apoptosis. Taken together, our findings suggest that sialyl-glycoconjugates present in cholesterol-rich microdomains form complexes with Hsc70 or Hsp70 that act as triggers for SBL/RC-RNase to induce apoptosis through a pathway involving the activation of caspase-3 and caspase-8.

13C-phenylalanine breath test detects altered phenylalanine kinetics in schizophrenia patients.

Phenylalanine is an essential amino acid required for the synthesis of catecholamines including dopamine. Altered levels of phenylalanine and its metabolites in blood and cerebrospinal fluid have been reported in schizophrenia patients. This study attempted to examine for the first time whether phenylalanine kinetics is altered in schizophrenia using L-[1-(13)C]phenylalanine breath test ((13)C-PBT). The subjects were 20 chronically medicated schizophrenia patients (DSM-IV) and the same number of age- and sex-matched controls. (13)C-phenylalanine (99 atom% (13)C; 100 mg) was administered orally and the breath (13)CO(2) /(12)CO(2) ratio was monitored for 120 min. The possible effect of antipsychotic medication (risperidone (RPD) or haloperidol (HPD) treatment for 21 days) on (13)C-PBT was examined in rats. Body weight (BW), age and diagnostic status were significant predictors of the area under the curve of the time course of Δ(13)CO(2) (‰) and the cumulative recovery rate (CRR) at 120 min. A repeated measures analysis of covariance controlled for age and BW revealed that the patterns of CRR change over time differed between the patients and controls and that Δ(13)CO(2) was lower in the patients than in the controls at all sampling time points during the 120 min test, with an overall significant difference between the two groups. Chronic administration of RPD or HPD had no significant effect on (13)C-PBT indices in rats. Our results suggest that (13)C-PBT is a novel laboratory test that can detect altered phenylalanine kinetics in chronic schizophrenia patients. Animal experiments suggest that the observed changes are unlikely to be attributable to antipsychotic medication.

Pharmacotherapeutic determinants for QTc interval prolongation in Japanese patients with mood disorder.

An increased incidence of sudden death has been observed among patients treated with antidepressants. A prolonged QTc interval is a known prognostic factor for fatal arrhythmia, and several studies have shown that the use of antidepressants can cause a prolonged QTc interval. However, few studies, especially in Japan, have compared the effects of multiple drugs on QTc interval or examined dose relationships in a clinical setting.We compared the effects of antidepressants on QT interval, corrected to QTc by Bazett's formula, in 729 Japanese patients who were diagnosed with mood disorder.Using stepwise multiple linear regression analysis, we found that the use of tricyclic antidepressants (P<0.01) and concomitant use of antipsychotics (P<0.05), as well as advanced age and being female (known factors for prolonged QTc interval; both P<0.01), significantly prolonged the QTc interval. Analysis of individual antidepressants also revealed that the use of clomipramine (P<0.01) and amitriptyline (P<0.05) significantly prolonged the QTc interval.Our results reveal that tricyclic antidepressants, especially clomipramine and amitriptyline, confer a risk of prolonged QTc interval in a dose-dependent manner. The selective serotonin reuptake inhibitors investigated (fluvoxamine, paroxetine, sertraline) were not indicated as risk factors for QTc prolongation.

Wideband spectral compression of wavelength-tunable ultrashort soliton pulse using comb-profile fiber.

We demonstrated spectral compression of ultrashort soliton pulses in a wide wavelength region based on an adiabatic soliton spectral compression technique using a comb-profile fiber. The comb-profile fiber was carefully designed using numerical analysis and fabricated using a conventional single-mode fiber and a dispersion-shifted fiber. The spectral width of a 200 fs soliton pulse was compressed from 12 to 15 nm to 0.54-0.71 nm in the wavelength region 1620-1850 nm, giving a spectral compression factor of up to 19.8-25.9. Owing to the soliton effect, the side lobe level was suppressed to -19.2 to -9.7 dB.

Production of active MMP7 in E. coli and its application for metalloproteinase inhibitors screening.

MMP-7 is the smallest metalloproteinase. Its unregulated activities and existence in serum are recently known to be tightly related with life-threatening disease such as cardiac disease and several cancers. The protein production is thought to be useful for its characterization and antibody generation. Although many attempts at bacterial expressions have been conducted, they were recovered as insoluble and inactive protein. In this study, after soluble expression, single-step purification and conversion to active protease, it was applied for the screening secretory metalloproteinase inhibitors in conditioned media of human cancer cells.

Purification and biochemical characterization of a D-galactose binding lectin from Japanese sea hare (Aplysia kurodai) eggs.

A lectin was purified from Japanese sea hare Aplysia kurodai by lactosyl-agarose affinity chromatography. The molecular mass of the lectin was determined to be 56 and 32 kDa by SDS-PAGE under non-reducing and reducing conditions, respectively. It was found to agglutinate trypsinized and glutaraldehyde-fixed rabbit and human erythrocytes in the absence of divalent cations. The lectin exhibited stable thermo-tolerance as it retained hemagglutinating activity for 1 h even at 80 degrees C and showed stability at pH 10. By contrast, it was very sensitive at pH less than 5 and in the presence of the sulfhydryl-group preserving reagent, beta-mercaptoethanol. The hemagglutinating activity by the lectin was specifically inhibited by D-galactose, galacturonic acid, methyl-alpha- and methyl-beta-D-galactopyranoside, lactose, melibiose, and asialofetuin. The association rate constant (k(ass)) and dissociation rate constant (k(diss)) were determined for the lectin to be 4.3 x 10(5) M(-1) x sec(-1) and 2.2 x 10(-3) sec(-1), respectively, using a surface plasmon resonance biosensor. The lectin moderately inhibited cell proliferation in the P388 cell line dose dependently. Interestingly, lectin-treated cells did not show a fragmented DNA ladder as is caused by apoptosis, suggesting that the cell proliferation inhibition was caused by another unknown mechanism.

Serine racemase binds to PICK1: potential relevance to schizophrenia.

Accumulating evidence from both genetic and clinico-pharmacological studies suggests that D-serine, an endogenous coagonist to the NMDA subtype glutamate receptor, may be implicated in schizophrenia (SZ). Although an association of genes for D-serine degradation, such as D-amino acid oxidase and G72, has been reported, a role for D-serine in SZ has been unclear. In this study, we identify and characterize protein interacting with C-kinase (PICK1) as a protein interactor of the D-serine synthesizing enzyme, serine racemase (SR). The binding of endogenous PICK1 and SR requires the PDZ domain of PICK1. The gene coding for PICK1 is located at chromosome 22q13, a region frequently linked to SZ. In a case-control association study using well-characterized Japanese subjects, we observe an association of the PICK1 gene with SZ, which is more prominent in disorganized SZ. Our findings implicating PICK1 as a susceptibility gene for SZ are consistent with a role for D-serine in the disease.

Prognostic analysis of pulmonary adenocarcinoma subclassification with special consideration of papillary and bronchioloalveolar types.

AIMS: The third edition of the World Health Organization (WHO) classification of lung tumours has been published and is expected to become the standard nomenclature. The aim of this study was to assess the usability and prognostic significance of the WHO classification in comparison with other recent classifications. METHODS AND RESULTS: One hundred and forty-seven resected pulmonary adenocarcinoma cases were reviewed and histologically classified according to the WHO classification (1999) and the classification by Noguchi (1995). Papillary carcinomas as described by Silver and Askin (1997) were also identified. Since the papillary type in the WHO classification is not strictly defined, we compared the following two kinds of WHO classification: (i) WHO-N; WHO classification adopting Noguchi Type F as the definition of the papillary type, namely, pure papillary adenocarcinoma without a bronchioloalveolar component; (ii) WHO-SA; WHO classification adopting papillary carcinoma by Silver and Askin as the definition of the papillary type, namely, tumour with papillary structure constituting at least 75% of the lesion. The bronchioloalveolar carcinoma of the WHO classification showed a better prognosis than other subtypes in both overall and Stage I disease limited survival analysis. In analysis limited to Stage III disease, only the papillary type of WHO-SA showed a significantly worse prognosis. CONCLUSIONS: WHO-SA is recommended for prognostic correlation.

Survival of Tdc transposable elements of the En/Spm superfamily in the carrot genome.

Three subfamilies of the En/Spm-type transposable element of carrot, Tdc A, B, and C, were characterized. It was supposed that the Tdc A subfamily may include autonomous elements which can produce transposases. Tdc B elements are defective, but still generate transcripts containing mutant open reading frame (ORF) sequences for transposases. The single member of the Tdc C group recovered seems to be a pseudogene. The sequences of the transposase ORFs of Tdc A and Tdc B elements are more highly conserved than those of the 5; and 3; untranslated regions and introns, as is found in other structural genes that are subject to selection. These observations indicate that the mutations in the nucleotide sequences of the Tdc elements occurred in the host genome. However, the mutations in the 5; and 3; untranslated regions and introns, which may not be sufficient to prevent transposition, accumulated in autonomous elements, which could transpose and produce copies. When the reproduction rate and the rate of disabling mutations reached an equilibrium, that is, when the birth rate of the transposable elements in the genome equalled the death rate, the population of elements achieved a stationary state in the genome, and could thus survive.

Periodate-treated, non-anticoagulant heparin-carrying polystyrene (NAC-HCPS) affects angiogenesis and inhibits subcutaneous induced tumour growth and metastasis to the lung.

Periodate-treated, non-anticoagulant heparin-carrying polystyrene consists of about ten periodate-oxidized, alkaline-degraded low molecular weight-heparin chains linked to a polystyrene core and has a markedly lower anti-coagulant activity than heparin. In this study, we evaluated the effect of non-anticoagulant heparin-carrying polystyrene on tumour growth and metastasis. Non-anticoagulant heparin-carrying polystyrene has a higher activity to inhibit vascular endothelial growth factor-165-, fibroblast growth factor-2- or hepatocyte growth factor-induced human microvascular endothelial cell growth than heparin, ten periodate-oxidized-heparin and ten periodate-oxidized-low molecular weight-heparin, which is probably due to the heparin-clustering effect of non-anticoagulant heparin-carrying polystyrene. Non-anticoagulant heparin-carrying polystyrene inhibited human microvascular endothelial cell, B16 melanoma and Lewis lung cancer cell adhesion to Matrigel-coated plates. Non-anticoagulant heparin-carrying polystyrene also showed strong inhibitory activities in the tubular formation of endothelial cells on Matrigel and B16-melanoma and Lewis lung cancer cell invasion in a Matrigel-coated chamber assay. In vivo studies showed that growth of subcutaneous induced tumours and lung metastasis of B16-melanoma and Lewis lung cancer cells were more effectively inhibited by non-anticoagulant heparin-carrying polystyrene than ten periodate-oxidized-heparin and ten periodate-oxidized-low molecular weight-heparin. Furthermore, non-anticoagulant heparin-carrying polystyrene markedly reduced the number of CD34-positive vessels in subcutaneous Lewis lung cancer tumours, indicating a strong inhibition of angiogenesis. These results suggest that non-anticoagulant heparin-carrying polystyrene has an inhibitory activity on angiogenesis and tumour invasion and may be very useful in cancer therapy.

Expression of matrix metalloproteinases in invasive pulmonary adenocarcinoma with bronchioloalveolar component and atypical adenomatous hyperplasia.

To investigate the association between the expression of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) and the clinicopathological features in lepidic and invasive components of adenocarcinoma of the lung, we performed immunostaining for type IV collagen, various MMPs, and TIMPs in 27 cases of invasive adenocarcinomas and 5 cases of atypical adenomatous hyperplasia of alveolar epithelial cells (AAH). Mean extent of lepidic growth was 61% and the survival was significantly better in cases with 50% or more lepidic component. The preservation of type IV collagen in lepidic areas correlated inversely with lymphatic or vascular invasion (P = 0.02 and 0.002, respectively). Five-year survival was reduced in cases showing destruction of type IV collagen (P = 0.004) or expression of MMP-2 (P = 0.008) in lepidic areas. MMP-2 co-localized with MT-1-MMP (its activating enzyme) and TIMP-2 in neoplastic cells. Reactivity for other MMPs and TIMPs did not correlate with destruction of type IV collagen or prognosis. Type IV collagen was preserved in all cases of AAH. MMP-2, but not MT-1-MMP, was expressed in two of the five cases of AAH. Immunostaining for type IV collagen MMP-2 is useful in evaluating the prognosis of the lung.

Experimental evaluation of photocrosslinkable chitosan as a biologic adhesive with surgical applications.

BACKGROUND: In various surgical cases, effective tissue adhesives are required for both hemostasis (eg, intraoperative bleeding) and air sealing (eg, thoracic surgery). We have designed a chitosan molecule (Az-CH-LA) that can be photocrosslinked by ultraviolet (UV) light irradiation, thereby forming a hydrogel. The purpose of this work was to evaluate the effectiveness and safety of the photocrosslinkable chitosan hydrogel as an adhesive with surgical applications. METHODS: The sealing ability of the chitosan hydrogel, determined as a bursting pressure, was assessed with removed thoracic aorta, trachea, and lung of farm pigs and in a rabbit model. The carotid artery and lung of rabbits were punctured with a needle, and the chitosan hydrogel was applied to, respectively, stop the bleeding and the air leakage. In vivo chitosan degradability and biologic responses were histologically assessed in animal models. RESULTS: The bursting pressure of chitosan hydrogel (30 mg/mL) and fibrin glue, respectively, was 225 +/- 25 mm Hg (mean +/- SD) and 80 +/- 20 mm Hg in the thoracic aorta; 77 +/- 29 mm Hg and 48 +/- 21 mm Hg in the trachea; and in the lung, 51 +/- 11 mm Hg (chitosan hydrogel), 62 +/- 4 mm Hg (fibrin glue, rubbing method), and 12 +/- 2 mm Hg (fibrin glue, layer method). The sealing ability of the chitosan hydrogel was stronger than that of fibrin glue. All rabbits with a carotid artery (n = 8) or lung (n = 8) that was punctured with a needle and then sealed with chitosan hydrogel survived the 1-month observation period without any bleeding or air leakage from the puncture sites. Histologic examinations demonstrated that 30 days after application, a fraction of the chitosan hydrogel was phagocytosed by macrophages, had partially degraded, and had induced the formation of fibrous tissues around the hydrogel. CONCLUSIONS: A newly developed photocrosslinkable chitosan has demonstrated strong sealing ability and a great potential for use as an adhesive in surgical operations.

Computed tomography-guided frameless stereotactic radiotherapy for stage I non-small cell lung cancer: a 5-year experience.

PURPOSE: Stereotactic radiotherapy (SRT) is highly effective for brain metastases from non-small-cell lung cancers (NSCLCs). As such, primary lesions of NSCLC may also be treated effectively by similar focal high-dose SRT. METHODS AND MATERIALS: Between October 1994 and June 1999, 50 patients with pathologically proven T1-2N0 M0 NSCLC were treated by CT-guided frameless SRT. Of these, 21 patients were medically inoperable and the remainder were medically operable but refused surgery. In most patients, SRT was 50-60 Gy in 5-10 fractions for 1-2 weeks. Eighteen patients also received conventional radiotherapy of 40-60 Gy in 20-33 fractions before SRT. RESULTS: With a median follow-up period of 36 months (range 22-66), 30 patients were alive and disease free, 3 were alive with disease, 6 had died of disease, and 11 had died intercurrently. Local progression was not observed on follow-up CT scans in 47 (94%) of 50 patients. The 3-year overall survival rate was 66% in all 50 patients and 86% in the 29 medically operable patients. The 3-year cause-specific survival rate of all 50 patients was 88%. No definite adverse effects related to SRT were noted, except for 2 patients with a minor bone fracture and 6 patients with temporary pleural pain. CONCLUSIONS: SRT is a very safe and effective treatment for Stage I NSCLC. Additional studies involving a larger patient population and longer follow-up periods are warranted to assess this new treatment for early-stage lung cancer.

Estimation of anti-platelet drugs on human platelet aggregation with a novel whole blood aggregometer by a screen filtration pressure method.

1. The effects of anti-platelet drugs on human whole blood aggregation were evaluated using a novel whole blood aggregometer by a screen filtration pressure (SFP) method. 2. The SFP whole blood aggregometer was found to successfully detect whole blood aggregation induced by ADP, collagen and TRAP by measuring the SFP of blood samples. The platelet aggregation threshold index (PATI), the concentration of agonist required with an inducing pressure rate of 50%, varied time-dependently after collection of blood. High values for ADP and collagen were noted immediately after blood collection, suggesting low aggregation activity of platelets, and gradually increase thereafter. 3. Cilostazol (phosphodiesterase 3 inhibitor), dipyridamole, aspirin and tirofiban all inhibited whole blood aggregation in vitro. Inhibitory effects of cilostazol and dipyridamole, but not tirofiban, were markedly enhanced 6 or 7 fold by long pre-incubation (60 min), compared with short pre-incubation (2 min). Such enhancement was only observed with ADP- and not collagen-induced whole blood aggregation. A similar phenomenon was also observed for aggregation with platelet rich plasma (PRP). Cilostazol inhibition of ADP-induced platelet aggregation was more potent with PRP than whole blood (PATI(200)=3.80+/-0.95 microM for whole blood; 2.04+/-0.61 microM for PRP). Inhibitory effects of dipyridamole were attenuated in PRP without erythrocytes. 4. These results demonstrate that the SFP aggregometer can sensitively detect anti-platelet aggregatory effects of various kinds of drugs. So that it is a useful tool for evaluation of anti-platelet drugs.

Lung carcinoma with rhabdoid cells: a clinicopathological study and survival analysis of 14 cases.

AIMS: We determined the clinicopathological features of primary lung carcinomas with rhabdoid cells by defining the immunophenotype of rhabdoid cells and analysing survival. METHODS AND RESULTS: Rhabdoid cells are distinctive in having an eccentric nucleus and a large intracytoplasmic inclusion on routinely stained sections. Based on the number of rhabdoid cells, 45 cases of large cell carcinoma were divided into the following three types: lung tumour with a rhabdoid phenotype (LTRP) (n=4), lung carcinoma with a small number of rhabdoid cells (LCSR) (n=10), large cell carcinoma containing no rhabdoid cells (LCNR) (n=31). LTRP is composed of at least 10% rhabdoid cells. In LCSR the percentage of rhabdoid cells is less than 10%. LTRP and LCSR are associated with locally advanced disease. Immunohistochemical stains were positive for epithelial markers in all LTRP and eight LCSR, for neuroendocrine markers in one LTRP and three LCSR. The outcome is worse for patients with LTRP than LCSR or LCNR. LCSR shows a trend close to LCNR. Stage-matched survival analysis, however, revealed no statistically significant difference among the histological subtypes. CONCLUSIONS: Rhabdoid cells are heterogeneous except for epithelial markers and vimentin positivity. Less than 5% of rhabdoid cells has a negligible effect on prognosis.

Hypersensitivity of melatonin suppression in response to light in patients with delayed sleep phase syndrome.

Patients with delayed sleep phase syndrome (DSPS) experience a chronic mismatch between the usual daily schedule required by the individual's environment and their circadian sleep-wake pattern, resulting in major academic, work, and social problems. Although functional abnormalities of the circadian pacemaker system have been reported in patients with DSPS, the etiology of DSPS has not been fully elucidated. One hypothesis proposed to explain why patients with DSPS fail to synchronize their 24 h sleep-wake cycle to their environment is that they might have reduced sensitivity to environmental time cues, most notably light-dark cycles. Therefore, we compared the sensitivity of melatonin suppression in response to light in patients with DSPS and normal control subjects. Fifteen patients with DSPS and age- and sex-matched healthy controls were studied. As the melatonin secretion rhythm in patients with DSPS was expected to be delayed compared to the controls, the time of peak melatonin secretion was determined in each subject in the first session. In the second session, each subject was exposed to light with an intensity of 1000 lux for 2 h beginning 2 h prior to his or her peak melatonin secretion. Melatonin was measured by radioimmunoassay in saliva sampled every 30 minutes during the period of light exposure. Suppression of the melatonin concentration in saliva was dependent on duration of light exposure. In addition, the suppressive effect of light on the melatonin concentration was significantly greater in patients with DSPS than in control subjects. The results suggest hypersensitivity to nighttime light exposure in patients with this syndrome. Our findings therefore suggest that evening light restriction is important for preventing patients with DSPS from developing a sleep phase delay.

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.

Comparative study of blood group-recognizing lectins toward ABO blood group antigens on neoglycoproteins, glycoproteins and complex-type oligosaccharides.

Binding specificities of ABO blood group-recognizing lectins toward blood group antigens on neoglycoproteins, glycoproteins and complex-type oligosaccharides were studied by lectin-blotting analysis, enzyme linked immunosorbent assay and lectin-conjugated agarose column chromatography. Human serum albumin conjugated with A- and B-trisaccharides was clearly recognized by Helix pomatia (HPA), Phaseolus lunatus, Dolichos biflorus agglutinins, and Griffonia simplicifolia I agglutinin B(4), respectively. Almost the same results were obtained for human group A and B ovarian cyst and A-active hog gastric mucins, but Glycine max agglutinin only reacted to the group A hog mucin. When human plasma von Willebrand factor (vWF), having Asn-linked blood group antigens, was tested, HPA was highly sensitive to blood group A antigen on the vWF. Ulex europaeus agglutinin I (UEA-I) preferentially bound to the vWF from blood group O plasma. Within the GalNAc-recognizing lectins examined, a biantennary complex-type oligosaccharide having the blood group A structure retarded on an HPA-agarose column, and the affinity was diminished after digestion with alpha-N-acetylgalactosaminidase. This product bound to UEA-I agarose column. These results indicate that HPA and UEA-I are most sensitive for detection of glycoproteins possessing small amounts of blood group A and H antigens and also useful for fractionation of complex-type oligosaccharides with blood group A and H antigens, respectively.