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Fish collagen peptide (FCP) is a water-soluble polymer with easy accessibility, bioactivity, and reactivity due to its solubility. The gelation of FCP can be carried out by chemical crosslinking, but the mechanical strength of FCP hydrogel is very low because of its intrinsically low molecular weight. Therefore, the mechanical properties of FCP gel should be improved for its wider application as a biomaterial. In this study, we investigated the mechanical properties of M-FCP gel in the context of understanding the influence of chitin nanofibers (CHNFs) on FCP hydrogels. FCP with a number average molecular weight (Mn) of ca. 5000 was reacted with glycidyl methacrylate (GMA) and used for the preparation of photocrosslinked hydrogels. Subsequently, composite hydrogels of methacrylate-modified FCP (M-FCP) and CHNF were prepared by the photoirradiation of a solution of M-FCP containing dispersed CHNF at an intensity of ~60 mW/cm2 for 450 s in the presence of 2-hydroxy-1-[4-(hydroxyethoxy)phenyl]-2-methyl-1-propanone (Irgacure 2959) as a photoinitiator. Compression and tensile tests of the FCP hydrogels were carried out using a universal tester. The compression and tensile strength of the hydrogel increased 10-fold and 4-fold, respectively, by the addition of 0.6% CHNF (20% M-FCP), and Young’s modulus increased 2.5-fold (20% M-FCP). The highest compression strength of the M-FCP/CHNF hydrogel was ~300 kPa. Cell proliferation tests using fibroblast cells revealed that the hydrogel with CHNF showed good cell compatibility. The cells showed good adhesion on the M-FCP gel with CHNF, and the growth of fibroblast cells after 7 days was higher on the M-FCP/CHNF gel than on the M-FCP gel without CHNF. In conclusion, we found that CHNF improved the mechanical properties as well as the fibroblast cell compatibility, indicating that M-FCP hydrogels reinforced with CHNF are useful as scaffolds and wound-dressing materials.

α-Emission radiotherapeutics has potential to be one of most effective cancer therapeutics. Herein, we report a facile synthesis of an 211At-labeled immunoconjugate for use as an α-emission molecular targeting therapy. We synthesized a tetrazine probe modified with closo-decaborate(2-), a prosthetic group that forms a bioavailable stable complex with 211At. Our one-pot three-component double-click labeling method was used to attach decaborate to trastuzumab (anti-HER2 antibody) using decaborate-tetrazine and TCO-aldehyde probes without reducing the antibody binding affinity. Labeling the decaborate-attached trastuzumab with 211At produced in the cyclotron at the RIKEN Nishina Center, at which highly radioactive 211At can be produced, readily furnished the 211At-labeled trastuzumab with a maximum specific activity of 15 MBq μg−1 and retention of the native binding affinity. Intratumor injection of the 211At-labeled trastuzumab in BALB/c nude mice implanted with HER2-expressing epidermoid cancer cells yielded efficient accumulation at the targeted tumor site as well as effective suppression of tumor growth.

The aim of this study was to identify relevant Rf chemical species by using reversed-phase extraction chromatography with 2-thenoyltrifluoroacetone (TTA) resin as the stationary phase. Because TTA can be used to extract specific metal ions, the distribution ratios of the system enabled determination of the specific complex formation constant of Rf. We performed several experiments on chemical systems with Zr, Hf, No, and Rf, determined their adsorption coefficients, and deduced the K d values for Rf.

A new non-linearity reduction technique for stochastic flash ADC (SF-ADC) is proposed, focusing on distribution of comparator input-referred offsets. The SF-ADC test chip fabricated in a 130-nm CMOS process demonstrated the proposed technique can improve SNDR. In addition, the digital re-quantization also can improve the linearity more, where quantization level and fractional correction can be optimized using genetic algorithm.

Biocompatibility of cinnamoyl-modified carbohydrate materials is not well-known, while they are attracting attention as a photoreactive material. In order to investigate biocompatible properties of cinnamoyl-modified carbohydrate, hydroxypropyl cellulose (HPC) was reacted with cinnamoyl chloride to yield cinnamoyl-modified HPC (HPC-C) for a cell proliferation test. HPC-Cs with three different degrees of substitution (DS) were prepared by changing a feed ratio of cinnamoyl chloride to HPC. The DS of the products ranged from 1.3 to 3.0 per one hydroxylpropyl anhydroglucose unit. Thermal analysis using DSC and TGA showed that the HPC-C with higher DS has a glass transition temperature and higher thermal stability. Ultraviolet (UV) light was irradiated on the HPC-C thin films, and changes in the UV–vis spectrum of the films were examined. In the course of UV irradiation, the absorbance at 280 nm was reduced. Fibroblast cells were cultured on the photocrosslinked HPC-C films, and cell growth was examined. The cell proliferation test revealed that the photocrosslinked HPC-C films have good compatibility with fibroblast cells.

Trehalose cinnamoyl esters (TCs) were synthesized by esterification of trehlaose with cinnamoyl chloride. Two TCs with different degrees of substitution were synthesized, and trehalose cinnamoyl ester thin film-coated glass plates were prepared by a dip-coating method. Photocuring of the TCs was confirmed by ultraviolet–visible spectral changes. The surface of photo-cured TCs were found smooth as observed in a scanning electron microscope. The cell proliferation on the photo-cured TCs was investigated using 3T3 Swiss Albino mouse embryo fibroblasts. The results of the cell proliferation assay revealed that the photo-cured TCs with higher degrees of substitution promoted the cell proliferation compared with a polystyrene culture plate and the photo-cured TCs with lower degrees of substitution. The contact angle of the photo-cured TCs with higher degrees of substitution was 101.0° ± 1.6°, which is much higher than that of the polystyrene culture plate, and it is out of the range known to be suitable for cell adhesion. Nevertheless, the cell unexpectedly grew best on the photo-cured TCs with higher degrees of substitution. Fibronectin binding assay was carried out using fluorescent probe-modified fibronectin, and more fibronectin was found adsorbed onto photo-cured TCs than a polystyrene culture plate.

We assessed the stability of orthodontic mini-implants in young rats. Male rats with mean ages of 6 weeks (n = 16) and 20 weeks (n = 16) were divided into four groups (n = 8 each). In the 6- and 20-week immediate-loading groups, immediately after placement, mini-implants were exposed to an experimental traction force for 2 weeks. In the 6- and 20-week healing groups, the force was applied for 2 weeks after a 6-week healing period. Right tibiae served as the test limbs and the left tibiae as controls. A Periotest device was used to measure mini-implant mobility after traction, and Tukey's test was used to compare Periotest values among groups. The results showed significantly greater mobility in the 6-week immediate-loading group than in the 20-week imme- diate-loading and 6- and 20-week healing groups, and significantly less mobility in the 6-week healing group than in the 20-week immediate-loading group (P < 0.05). Mini-implants were stable during the healing period. The results indicate that mini-implants can be used for orthodontic anchorage in juvenile patients if the duration of healing is sufficient. (J Oral Sci 56, 113-118, 2014)

A photocrosslinkable trehalose derivative carrying mesogenic groups was synthesized by esterification reactions. The derivative (TC-HBPHA) was synthesized by the reaction of partially cinnamoyl-modified trehalose (TC4) with 4-(4-hexyloxybenzoyloxy)phenoxy-6-oxohexanoic acid (HBPHA) as a mesogenic unit. TC-HBPHA showed a nematic liquid crystalline mesophase at a temperature range from 150 °C to 175 °C in the heating process under observation with a polarized optical microscope. The dimerization of the cinnamoyl groups of TC-HBPHA by ultraviolet (UV) light irradiation was monitored by ultraviolet-visible (UV-Vis) spectroscopy and Fourier transform infrared (FT-IR) spectroscopy. The photocrosslinked film was obtained after the UV irradiation of TC-HBPHA, and it kept the liquid crystalline mesophase at almost the same temperature range. Fibroblast cells cultured on the photocrosslinked TC-HBPHA proliferated as well as on the polystyrene culture plate, indicating that the film has no toxicity. Interestingly, some cells on photocrosslinked TC-HBPHA had a spindle shape and aligned characteristically.

We examined the receptor-mediated effects of extracellular ATP on neuronal differentiation of PC12 cells, Neuro2a cells and MEB5 cells by using a series of receptor antagonists. The P2Y13 receptor antagonist MRS2211 significantly accelerated neurite outgrowth in all cases. Treatment with nerve growth factor (NGF) alone activated ERK1/2 in PC12 cells, and the activation was further increased by MRS2211. These results suggest involvement of P2Y13 receptor in suppression of neuronal differentiation. Thus, P2Y13 receptor antagonists might be candidates for treatment of neurodegenerative diseases.

The biomechanical influences of primary factors on titanium mini-implant, which is used as an anchorage for orthodontic tooth movement, were quantified using the three-dimensional finite element method. Six types of finite element models were designed to show various thread pitches from 0.5 to 1.5 mm. Three models were designed with abutment and three other models without abutment. A traction force of 2 N was applied to the head of the mini-implant or abutment to be at 45 degrees to the bone surface. No remarkable differences were observed in the stress distribution patterns regardless of thread pitch variance. However, the stress distribution was remarkably different between models with abutment and without abutment. The maximum stress of the model with abutment and thread pitch 0.5 mm was the least as compared with the other models. Areas of high-level stress were obviously smaller than in the models without abutment. The plots of the displacement distributions of the models with abutment also presented significant pattern differences as compared with the models without abutment. The high-level area was localized to the head of the implant and the abutment in models with abutment. Therefore, the existence of the abutment is significantly useful in decreasing the stress concentration on the bone, while the effect of thread pitch was uncertain.

Photocrosslinked hydrogels reinforced by microfibrillated cellulose (MFC) were prepared from a methacrylate-functionalized fish elastin polypeptide and MFC dispersed in dimethylsulfoxide (DMSO). First, a water-soluble elastin peptide with a molecular weight of ca. 500 g/mol from the fish bulbus arteriosus was polymerized by N,N′-dicyclohexylcarbodiimide (DCC), a condensation reagent, and then modified with 2-isocyanatoethyl methacrylate (MOI) to yield a photocrosslinkable fish elastin polypeptide. The product was dissolved in DMSO and irradiated with UV light in the presence of a radical photoinitiator. We obtained hydrogels successfully by substitution of DMSO with water. The composite gel with MFC was prepared by UV irradiation of the photocrosslinkable elastin polypeptide mixed with dispersed MFC in DMSO, followed by substitution of DMSO with water. The tensile test of the composite gels revealed that the addition of MFC improved the tensile properties, and the shape of the stress–strain curve of the composite gel became more similar to the typical shape of an elastic material with an increase of MFC content. The rheology measurement showed that the elastic modulus of the composite gel increased with an increase of MFC content. The cell proliferation test on the composite gel showed no toxicity.

We compared the antithrombotic effect of anti-GPIIb/IIIa antibody Fab fragment YM337 with that of a thromboxane A2 synthetase inhibitor, sodium ozagrel. With the monkeys under halothane anesthesia, the right middle cerebral artery was observed via a transorbital approach without cutting the dura mater. Photoillumination (wavelength 540 nm) was applied to the middle cerebral artery, and then rose bengal (20 mg kg(-1)) was administered intravenously. The experimental drugs were intravenously injected 15 min before rose bengal injection and followed by continuous infusion for 3 h after dye injection. The thrombotic occlusion induced by this photochemical reaction in monkey middle cerebral artery was reproducible. YM337 significantly prolonged the time to first occlusion and the total time of arterial patency during the 3-h observation period after dye injection. In contrast, sodium ozagrel had no significant effect. YM337 but not sodium ozagrel significantly inhibited ex vivo ADP-induced platelet aggregation. However, while sodium ozagrel significantly inhibited the thromboxane B2 generation accompanying arachidonic acid-induced platelet aggregation, YM337 had no effect on this variable. Neurological deficit in the YM337-treated animals was significantly milder than that in the control group. The area of infarct in the YM337 treatment animals was smaller than that in the control group. The novel selective GPIIb/IIIa antagonist YM337 was effective in ameliorating the decrease in patency of the middle cerebral artery and reducing the area of cerebral infarction in monkeys.

1. 1. The potential antiplatelet agent use of the whole molecule, F(ab) 2 and Fab fragments of humanized antiglycoprotein (GP) IIb/IIIa monoclonal antibody, hC4G1, were investigated in rhesus monkeys. 2. 2. Fab completely inhibited platelet aggregation 1 hr after an IV bolus administration of 1 mg/kg without a decrease in platelet count or prolongation of bleeding time, and the duration of inhibition was much shorter than that of F(ab) 2 3. 3. These results suggest that the Fab fragment of hC4G1 may be a more useful antiplatelet agent in patients with acute thromboembolic diseases than the whole molecule or F(ab) 2 fragments.

We have developed two monoclonal antibodies (MAbs), B6A3 and C4G1. The whole molecules of the two MAbs inhibited in vitro human platelet aggregation induced by either ADP, collagen or thrombin, and their F(ab')2 fragments inhibited ex vivo platelet aggregation induced by ADP in monkey. The concentrations necessary for complete inhibition were 5 and 1 microgram/ml for B6A3 and C4G1, respectively. The Fab fragment of C4G1 but not B6A3 inhibited platelet aggregation. B6A3 and C4G1 bound to activated platelets with dissociation constants of 0.25 and 0.82 nM, respectively. B6A3 recognized an epitope on beta 3, which was sensitive to reduction and alkylation of cystine residues, and C4G1 recognized a conformational epitope on the alpha IIb beta 3 complex, which was sensitive to EDTA. The binding of fibrinogen to activated platelets was inhibited by both MAbs. However, the binding of fibrinogen to isolated alpha IIb beta 3 was inhibited by the whole molecule of C4G1 but not B6A3, although both MAbs bound to the isolated alpha IIb beta 3. The binding of these MAbs to the isolated alpha IIb beta 3 was not inhibited by either Arg Gly Asp Ser (RGDS) or fibrinogen gamma-peptide. In addition, B6A3 but not C4G1 bound to human endothelial cells. These MAbs should contribute to the elucidation of the mechanism of platelet aggregation.

One notable complication of mini-implants that are used to provide anchorage in orthodontic treatment is loosening. The aim of this study was to evaluate the relationship between mini-implant mobility during the healing phase and the prognosis for implant stability. Twenty male Wistar rats (aged 20 weeks) were used. Drills with diameters of 0.8, 0.9, 1.0, and 1.1 mm were used to make pilot holes in the rat tibiae. The inserted mini-implants (diameter 1.4 mm; spearhead 1.2 mm; halfway between maximum and minimum 1.3 mm; length 4.0 mm) were subjected to an experimental traction of force for 3 weeks. Bone-to-implant contact (BIC) was observed histologically. Another 20 male rats (aged 20 weeks) underwent an identical procedure, and the stability of the mini-implants was measured using the Periotest before and after traction. The data were statistically analysed using Scheffé's test. The BIC ratios of the 0.9 and 1.0 mm groups were significantly greater than those of the other groups. The Periotest values measured 3 weeks after implant insertion were significantly lower (P0.05) than those measured at insertion, except in the 1.1 mm group. To obtain mini-implant stability, the hole diameter should be between 69 and 77 per cent of the diameter of the mini-implant. A significant decrease in the mobility of the mini-implants 3 weeks post-insertion implies a good prognosis for the subsequent mini-implant stability.

The aim of this study was to investigate the initial stability of tapered orthodontic miniscrews (T-type screws) after placement, the necessity of a healing period, and the propriety of immediate loading. Twenty male Wistar rats with a mean age of 20 weeks were divided into two groups. In the immediate-loading groups, straight orthodontic miniscrews (S-type screws) and T-type screws (five rats each) underwent experimental traction force for 2 weeks (W) immediately after placement. In the healing groups (S- and T-type, five rats each), force was applied for 2 W after a 6-W healing period. The right tibia in each rat was identified as the test limb, while the left tibia in each rat was used as the control group, and underwent no experimental force during the experimental period. The screw-to-bone contact was observed histologically and the bone-screw contact ratio was calculated. Scheffe's test was performed to compare the bone-screw contact ratio in each group using statistical software package (SPSS 8.0 for Windows). In the control group, the bone-screw contact ratio improved from 34.8 +/- 16.0 to 74.8 +/- 12.0 per cent with S-type screws in proportion to the experimental period (2 to 8 W, respectively). With the T-type screws in the test group, there was no significant difference between the immediate-loading and healing groups. In the immediate-loading group, the bone-screw contact ratio with T-type screws was significantly greater (82.3 +/- 15.0 per cent) than with the S-type screws (33.3 +/- 11.8 per cent; P < 0.05), suggesting that T-type screws can be used for orthodontic anchorage immediately after placement.

In recent years, mandibular expansion has been increasingly performed in conjunction with orthodontic treatment. Lateral tipping of the molars associated with mandibular expansion should, however, be considered, because excessive expansion may result in excessive buccal tooth inclination, which may disturb the occlusal relationship. This study was conducted to quantitatively clarify molar movement during mandibular expansion using the Schwarz appliance to determine the permissible limit of mandibular expansion as a clinical index for inclination movement. Inclinations in the masticatory surface of the first molar and intermolar width were measured before expansion (T1), after expansion (T2), and before edgewise treatment (T3). Lower plaster models from 29 subjects treated with expansion plates were used and compared with models from 11 control subjects with normal occlusion. The average treatment change (T1-T2) in intermolar width was 5.42 mm (standard deviation 1.98), and the average angle of buccal tooth inclination was 10.16 degrees (standard deviation 3.83). No significant correlation was found between age prior to treatment and the treatment period when they were compared with the intermolar width increments and inclination angles. There was a significant positive correlation between retention duration and the amount of expansion. The regression coefficient of the angle of buccal tooth inclination during expansion to the increment of the intermolar width was approximately 0.2. This means that 1 mm of expansion is accompanied by 5 degrees of molar lateral tipping. This coefficient is clinically useful for estimating the permissible limit for mandibular expansion.

The effects of YM337, the Fab fragment of a humanized anti-glycoprotein IIb/IIIa (GPIIb/IIIa) monoclonal antibody C4G1, on in vitro platelet function and binding properties were compared with those of abciximab, the Fab fragment of the human/murine chimeric anti-GPIIb/IIIa monoclonal antibody 7E3. Both agents completely inhibited platelet aggregation caused by all agonists tested except ristocetin. Further, both inhibited human platelet adhesion to von Willebrand factor, fibrinogen, fibronectin and subendothelial matrix with similar potency. Fibrinogen binding to washed platelets was dose-dependently inhibited by both agents. In binding assay using 125I-YM337 and 125I-abciximab, Kd values determined with platelet-rich plasma were 6.74 +/- 0.56 nM for YM337 and 6.65 +/- 1.45 nM for abciximab, and the number of binding sites were 42,700 +/- 3,000 for YM337 and 76,000 +/- 5,400 for abciximab. GPIIb/IIIa was precipitated from the solubilized fraction of platelets by both agents. In contrast, integrin alphavbeta3 was precipitated from the solubilized fraction of human umbilical vein endothelial cells by abciximab but not by YM337. Fibrinogen binding to purified GPIIb/IIIa was dose-dependently inhibited by both agents. In contrast, vitronectin binding to purified integrin alphavbeta3 was dose-dependently inhibited by abciximab but not by YM337, supporting the idea that abciximab reacts to integrin alphavbeta3. Therefore, YM337 was suggested to bind to a different epitope of GPIIb/IIIa from abciximab. These results suggest that YM337 specifically acts on platelet GPIIb/IIIa receptors and has similar inhibitory properties on platelet aggregation and platelet adhesion to abciximab.

SummaryWe examined the effect of a humanized anti-glycoprotein IIb/IIIa monoclonal antibody, YM337, on thrombolysis with tissue-type plasminogen activator in a copper coil-induced coronary thrombosis model in rhesus monkeys. Fifty minutes after the formation of an occlusive thrombus, a test drug was administered by either i.v. bolus injection followed by continuous infusion (YM337, 0.25 mg/kg + 1.5 μg/kg/min) or i.v. bolus injection (aspirin, 17 mg/kg). Sixty minutes after induction of the occlusive thrombus, thrombolysis was initiated with tPA at a total dose of 0.5 mg/kg intravenously administered over 60 min, with 10% given as an initial bolus. The median time to reperfusion was significantly shortened by YM337 [saline, 60 min (n = 5); aspirin, 45 min (n = 5); YM337, 30 min (n = 5)]. The incidence of reocclusion was significantly decreased by YM337 (saline, 4/4; aspirin, 5/5; YM337, 1/5), and the median time to reocclusion was significantly prolonged by YM337 [saline, 30 min (n = 4); aspirin, 30 min (n = 5); YM337, 180 min (n = 5)]. YM337 significantly reduced the thrombus protein content at the end of experiment. ADP-induced platelet aggregation was completely inhibited by YM337. These results suggest that YM337 may be of clinical value as an adjunctive agent in thrombolytic therapy for patients with acute myocardial infarction.

We directly compared the effects of YM337, the Fab fragment of the humanized monoclonal antibody C4G1, on platelet aggregation and template bleeding time with those of abciximab, the Fab fragment of the human/murine chimeric monoclonal antibody 7E3, in rhesus monkeys. The duration of inhibition of platelet aggregation by abciximab after i.v. bolus injection was much longer than that by YM337. Although YM337 significantly prolonged template bleeding time at 5 min after i.v. bolus injection, this action recovered within 1 h after injection. In contrast, although abciximab also prolonged template bleeding time, the duration of this effect was sustained. In a dose-escalating continuous infusion study, we evaluated the relationship between inhibition of platelet aggregation and prolongation of template bleeding time. Platelet aggregation was inhibited by over 80% by both agents at 3 microg/kg per min, and template bleeding time was prolonged to about 30 min at 30 microg/kg per min for YM337 and 10 microg/kg per min for abciximab. Interestingly, plasma concentrations between inhibition of platelet aggregation and prolongation of template bleeding time did not overlap with YM337, but did overlap with abciximab. These results suggest that YM337 allows easier control of antiplatelet activity with less effect on bleeding time than abciximab, and has a wider therapeutic window than abciximab.

SummaryThe antiplatelet and antithrombotic effects of the Fab fragment of the humanized antiplatelet glycoprotein (GP) IIb/IIIa monoclonal antibody C4G1 (YM337) were investigated in monkeys. First, the relationship between the inhibition of platelet aggregation and the prolongation of bleeding time was studied in rhesus monkeys. YM337 dose-dependently inhibited ex vivo platelet aggregation, with complete inhibition at doses higher than 0.25 mg/kg intravenous injection or 1.5 μg/kg/min infusion. At 0.25 mg/kg bolus injection followed by 1.5 μg/kg/min infusion, YM337 immediately and continuously inhibited platelet aggregation during the 6-h infusion period with platelet aggregation rapidly returning to over 50% of baseline within 1 h after the cessation of infusion. Template-bleeding time was significantly prolonged during the period of complete inhibition of platelet aggregation.Second, the antithrombotic effects of YM337 were investigated in a photochemically-induced thrombosis model in squirrel monkeys. YM337 at a dose of 1 mg/kg intravenous injection followed by 6 μg/kg/min infusion for 60 min prevented occlusive thrombus formation in all 4 monkeys. In contrast, time to occlusive thrombus formation did not change on intravenous bolus injection of aspirin 17 mg/kg (11.3 ± 5.2 min) or sodium ozagrel (9.4 ± 3.0 min) compared with saline (13.3 ± 4.0 min). YM337 but not aspirin or sodium ozagrel significantly inhibited ex vivo ADP-induced platelet aggregation, while all drugs completely inhibited arachidonic acid-induced platelet aggregation. However, while aspirin and sodium ozagrel inhibited the thromboxane B2 generation accompanying arachidonic acid-induced platelet aggregation, YM337 had no effect on this variable. Platelet counts and bleeding time showed no significant change in any group in this squirrel monkey model.These results indicate that YM337, with a short half-life, may be a useful therapeutic agent in patients with thrombotic disorders.

A humanized monoclonal IgG1 antibody, designated hC4G1, recognizes the fibrinogen receptor glycoprotein (GP)IIb/IIIa on platelets and inhibits platelet aggregation. When the F(ab')2 fragment of hC4G1 (F(ab')2 hC4G1) was administered to cynomolgus monkeys, all the monkeys showed inhibition of platelet aggregation ex vivo. Unexpectedly, a significant decrease in platelet count was observed in 5 of 18 monkeys. Antibodies against F(ab')2 hC4G1 were detected in the plasma of these monkeys by ELISA. Antibody activity in the plasma of these monkeys was significantly correlated with the intensity of platelet decrease (r = 0.84). The natural monkey antibodies to F(ab')2 hC4G1 were directed against the C-terminal region of F(ab')2 fragment common to all human and humanized IgG antibodies. Natural homo-reactive antibodies were also detected in human plasma from 15 of 40 healthy volunteers. Specificity was closely similar to that of the monkey antibodies. Affinity-purified human homoreactive antibodies enhanced phagocytosis of platelets treated with the F(ab')2 hC4G1. Monkey plasma with high homo-reactive antibody activity was confirmed to decrease platelet count when administered together with F(ab')2 hC4G1 to a monkey with low antibody activity. These results suggest that F(ab')2 of humanized and human antibodies causes elimination of the corresponding antigens from the circulation by homo-reactive antibodies.

We investigated the antithrombotic effects of a F(ab')2 fragment of a humanized anti-platelet glycoprotein (GP) IIb/IIIa monoclonal antibody, YM207, in a platelet-rich thrombosis model. Thrombus was induced in a mesenteric venule of squirrel monkeys by irradiation with filtered light in combination with i.v. administration of a fluorescent dye (sodium fluorescein). Time to occlusive thrombus formation was significantly prolonged by i.v. bolus injection of 0.3 mg/kg of YM207. At doses of 1 mg/kg and 3 mg/kg, complete occlusion did not occur during 60 min of the observation. Platelet aggregation in platelet-rich plasma was inhibited by YM207 at doses 0.3 mg/kg or more. A good correlation was obtained between inhibition of platelet aggregation and the percentage of GPIIb/IIIa receptors blocked by YM207. Bleeding time was significantly prolonged at a dose of 3 mg/kg. Platelet counts showed no significant change. These results suggest that YM207 is effective in preventing platelet-rich thrombus formation without inducing the prolongation of bleeding time or a significant decrease in platelet count. YM207 may be a useful therapeutic agent for the treatment of thrombotic disorders.

Two well-known antifungals, amphotericin B (AmB) and amphodinol 3 (AM3), are thought to exert antifungal activity by forming ion-permeable channels or pores together with sterol molecules. However, detailed molecular recognitions for AmB-sterol and AM3-sterol in lipid bilayers have yet to be determined. Toward (19)F NMR-based investigation of the molecular recognition underlying their potent antifungal activity, we synthesized 6-fluoro-ergosterol in five steps via ring opening of (5α,6α)-epoxide of ergosterol acetate with using novel combination of TiF(4) and n-Bu(4)N(+)Ph(3)SiF(2)(-). Then we evaluated its activity of promoting pore formation of AmB and AM3, and found that pore formation of AmB was barely promoted by 6-F-ergosterol in clear contrast to the dramatic promotion effect of unmodified ergosterol, whereas AM3 activity was markedly enhanced in the presence of 6-F-ergosterol, which was comparable to that of unmodified ergosterol. These results indicate that the introduction of an F atom at C6 position of ergosterol plays an inhibitory role in interacting with AmB, but it is not the case with AM3.

Our recent studies have shown that chick embryo epiphyseal cartilage synthesizes three distinct species of proteoglycan (PG-H, PG-Lb, and PG-Lt) which are analogous in having glycosaminoglycan side chains of the chondroitin (dermatan) sulfate type but different from one another in regard to the structure of core protein. In the present report, the expression of PG-H and PG-Lb has been studied in developing chick hind limbs (stages 19-33), using antibodies specific for these substances in indirect immunofluorescence. At the onset of cartilage morphogenesis (stage 24), PG-H became recognizable in the cartilage primordia, whereas a parallel section stained for PG-Lb showed no reaction. The first evidence of PG-Lb appearance was seen in a stage 28 cartilage (e.g., tibia) in which the cells in the middiaphysis became elongated in a direction perpendicular to the long axis of the cartilage. The PG-Lb fluorescence was confined to the zone of these flattened, disc-like cells, whereas the fluorescence for PG-H was uniformly distributed throughout the cartilage. With further development of cartilage (stage 29 approximately), the zone of flattened cells spread proximally and distally, and simultaneously large hypertrophied cells appeared at the diaphyseal region. During these zonal changes of cell morphology, the PG-Lb fluorescence remained restricted to the zone of flattened cells. Parallel sections stained for PG-H, in contrast, showed an evenly distributed pattern of the PG-H fluorescence throughout the cartilage. The results indicate that the appearance of PG-Lb is closely associated with the zonal changes of cell shape and orientation along the proximal-distal axis of the developing limb cartilage, and further suggest that the flattened chondrocytes in this particular zone have undergone additional changes in gene expression to form an extracellular matrix of still another chemical property.

Human erythrocytes contain a membrane protein, MACIF, which inhibits the formation of a membrane attack complex (MAC) of complement. We have cloned and sequenced the complementary DNA of MACIF messenger RNA. The amino acid sequence predicted from its nucleotide sequence consists of 128 amino acids. The amino-terminal 25 residues may correspond to a signal peptide. The carboxy-terminal sequence confirmed that MACIF is a glycosylphosphatidylinositol (GPI)-anchored protein. The amino acid sequence of MACIF was partially determined by established techniques for protein chemistry and the resultant sequence was consistent with that predicted from the nucleotide sequence. The results of sequence analyses also suggested that asparagine at the 18th position was N-glycosylated. When mRNA obtained from the MACIF cDNA clone with SP6 RNA polymerase was microinjected into Xenopus oocytes, the oocytes synthesized a product which exhibited MACIF activity and reacted with anti-MACIF antibody. Comparison of the predicted sequence revealed significant homology with mouse Ly-6 antigens.