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Background: Plasminogen activator inhibitor‐1 (PAI‐1) is the primary physiological regulator of urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA) activity. A number of studies have shown that elevated levels of PAI‐1 are related to pathological states such as an increased risk of arterial thrombotic events and a poor prognosis for cancer patients; however, there are few reports about PAI‐1 deficiency in humans because the disorder is very rare. Objective: To understand the in vivoimpact of a complete PAI‐1 deficiency, Serpine1−/−mice were generated; a number of in vivostudies have been conducted to elucidate the function of PAI‐1 using Serpine1−/−mice. The phenotypes demonstrated in Serpine1−/−mice, however, were quite different from those in humans. Therefore, it is necessary to find out and analyze SERPINE1deficiency in humans. Patient and methods: The patient is a 47‐year‐old woman who has had multiple episodes of major bleeding. Although most of the patient’s blood coagulation factors were functionally normal, her PAI‐1 antigen levels were undetectable. Therefore, DNA sequencing of the SERPINE1gene were analyzed. Results: The proband had a homozygous 1‐bp duplication (C) at exon 3 (c.356dupC; p.Ile120AspfsX42). Both wild‐type PAI‐1 (42.7 kDa) and mutated (Mut) PAI‐1 (14.7 kDa) were expressed in COS‐1 cells, although the level of Mut PAI‐1 expressed in the cell lysates was much lower. Wild‐type PAI‐1 was observed in the culture supernatant, whereas no Mut PAI‐1 was detected in the supernatant. Conclusions: Considering the results of the present study, the translation of mouse studies to humans must be performed with great care.

Functionalizing nanotubes: An easy method is developed to functionalize boron nitride nanotubes. Hydrogen peroxide is used to oxidize the surface of pristine BN nanotubes to get hydroxylated BN nanotubes, which are chemically active for further functionalization. The Figure shows hydroxylated BN nanotubes can be well dispersed in water (left), while pristine BN nanotubes are hydrophobic (right).

We report the results of stochastic molecular dynamics simulations for the structural formation of charged dendrimers and dendrimer-based star polymers. The variation in the radius of gyration explains quite well recent small-angle neutron-scattering experiments for higher-generation charged dendrimers under different pH conditions. We also clarify the properties of effective interaction between dendrimers for weak and strong electrostatic couplings in an aqueous solution.

Link protein (LP), an extracellular matrix protein in cartilage, stabilizes aggregates of hyaluronic acid (HA) and proteoglycans, including aggrecan and inter-alpha-trypsin inhibitor (ITI). We have shown previously that cartilage LP is present in the maturing rat and mouse ovary. In the present study, we have employed immunohistochemistry to examine the anatomical distribution of cartilage LP in the human ovary. The expression of cartilage LP was selectively detected in the cells within the granulosa compartment of the preovulatory dominant follicle. The HA-positive granulosa-lutein cells were found to be a cartilage LP-positive subpopulation. We subsequently studied the in vitro expression of cartilage LP in cultured human granulosa-lutein cells obtained at oocyte retrieval for in vitro fertilization. Analysis of cultured cells by enzyme-linked immunoaffinity assay, Western blotting and immunofluorescence microscopy revealed that gonadotropin stimulates cartilage LP production. Time-course studies indicated that the cartilage LP production was induced as early as with gonadotropin stimulation for 2 h, and the effect was sustained up to 8 h. Western blot analysis further revealed the presence of the macroaggregates composed of HA, ITI and cartilage LP in the gonadotropin-stimulated granulosa-lutein cell extracts. Collectively, the present results raise the possibility that cartilage LP forms extracellular structures that may have a regulatory function in the developing follicle in the human ovary.

¹³C and ¹²⁹Xe high-pressure (HP) magic-angle spinning (MAS) NMR was applied to a polystyrene/poly(vinyl methyl ether) (PS/PVME) = 50/50 blend under Xe pressure range of 0.0−4.0 MPa for investigating effects of the dissolved Xe atoms on the segmental motions of the individual component polymers. It is shown that the chemical shift of ¹²⁹Xe dissolved in the blend moves to the high-frequency side and the line becomes narrow with increasing Xe gas pressure. The ¹²⁹Xe higher-frequency shift is explained in terms of an increase of the Xe concentration in the blend. The line narrowing suggests that the Xe atomic diffusion is enhanced with increasing Xe concentration in the blend. On the other hand, the ¹³C line widths under MAS and ¹H dipolar decoupling (DD) evidently show that the dissolved Xe atoms enhance the segmental motions of the individual component polymers (plasticization effect) at Xe gas pressures higher than 1.0 MPa. Furthermore, the observed pressure and temperature dependence of the line widths demonstrates that the motional frequency difference between PS and PVME at high pressures is less than that observed at high temperatures. It is concluded that the dissolved Xe atoms not only enhance the segmental motions of PS and PVME but also work to reduce the motional heterogeneity in the blend.

Furosemide (frusemide) is a weakly acidic diuretic drug. Its absorption is poor and variable, in part due to its restricted sites of absorption, mainly the stomach. The narrow absorption window of this drug can be explained by pH partition theory. The purpose of this study was to investigate the feasibility of widening the absorption window of furosemide by controlling the pH in distal portions of the gastrointestinal tract with officially used additives. Methacrylate copolymer (Eudragit L100–55), hydroxypropylmethylcellulose phthalate (HP‐55) and hydroxypropylmethylcellulose acetate succinate (AS‐MF) were selected as additives. The pH of suspensions of these additives was about 4, and the pH was adjusted to about 6–7 by the addition of NaOH. The Eudragit L100–55 suspension was found to be the most resistant to NaOH titration. When Eudragit L100–55 was used in an in‐situ ileal loop experiment in rats, the pH of the intestinal contents was significantly reduced, from 7.9 ± 0.1 to 5.7 ± 0.1, and the plasma concentration of furosemide 15 min after administration was about 3 times higher than that in controls, 1.81 ± 0.42 μg mL−1vs 0.63 ± 0.08 μg mL−1. However, the plasma concentration of [14C] mannitol was not changed by the co‐administration of Eudragit L100–55. Furthermore, the AUC of furosemide was significantly increased by a factor of about 1.6 relative to that in controls by the co‐administration of Eudragit L100–55, to 21.4 ± 4.0 μg h mL−1from 13.3 ± 3.9 μg h mL−1, and the gastrointestinal pH in the midgut and ileum was significantly reduced, with most of the furosemide remaining in these segments at 2 h following the oral administration of furosemide with Eudragit L100–55 to rats. These findings clearly demonstrate that the addition of Eudragit L100–55 can increase the absorption of furosemide in distal portions of the gastrointestinal tract. In conclusion, it is feasible to widen the absorption window of furosemide by controlling the pH in distal portions of the gastrointestinal tract by the co‐administration of Eudragit L100–55.

Urinary trypsin inhibitor (UTI) forms membrane complexes with UTI-binding proteins (UTI-BPs) and initiates modulation of urokinase-type plasminogen activator (uPA) expression, which results in UTI-mediated suppression of cell invasiveness. It has been established that suppression of uPA expression and invasiveness by UTI is mediated through inhibition of protein kinase C-dependent signaling pathways and that human chondrosarcoma cell line HCS-2/8 expresses two types of UTI-BPs; a 40-kDa UTI-BP (UTI-BP(40)), which is identical to link protein (LP), and a 45-kDa UTI-BP (UTI-BP(45)). Here we characterize binding properties of UTI-BPs.UTI complexes in the cells. In vitro ligand blot, cell binding and competition assays, and Scatchard analyses demonstrate that both UTI-BP(40) and UTI-BP(45) bind (125)I-UTI. A deglycosylated form of UTI (NG-UTI), from which the chondroitin-sulfate side chain has been removed, binds only to UTI-BP(40). Additional experiments, using various reagents to block binding of (125)I-UTI and NG-UTI to the UTI-BP(40) and UTI-BP(45) confirm that the chondroitin sulfate side chain of UTI is required for its binding to UTI-BP(45). Analysis of binding of (125)I-UTI and NG-UTI to the cells suggests that low affinity binding sites are the UTI-BP(40) (which can bind NG-UTI), and the high affinity sites are the UTI-BP(45). In addition, UTI-induced suppression of phorbol ester stimulated up-regulation of uPA is inhibited by reagents that were shown to prevent binding of UTI to the 40- and 45-kDa proteins. We conclude that UTI must bind to both of the UTI-BPs to suppress uPA up-regulation.

We examined the relationships among the clinical efficacies of risperidone, plasma concentrations of risperidone and its active metabolite, 9hydroxyrisperidone, and changes in plasma free MHPG pMHPG in 14 schizophrenic patients. Clinical improvement in negative symptoms of schizophrenia treated with risperidone has been associated with increased pMHPG and, in the present study, there were positive correlations between plasma 9hydroxyrisperidone concentrations and increased pMHPG levels. These results suggest that risperidone might improve negative symptoms in schizophrenia by influencing noradrenergic neurons.

We analysed the early implantation tissues of normal women and of a patient with congenital factor XIII deficiency in order to study the role of maternal subunit A of factor XIII (XIIIA) in the development of extravillous cytotrophoblast. The patient had received adequate administration of factor XIIIA concentrate only up to 7 weeks of gestation (wG). Her pregnancy was maintained until the latter half of 8wG, but was terminated by intrauterine fetal death at 9wG. Immunohistochemical staining of cytokeratin, XIIIA and subunit S of factor XIII was performed in the early implantation tissues of normal women and of this patient. Numerous well-formed cytotrophoblastic shells and Nitabuch's layers were detected in implantation tissues at 7–8wG in normal women, and XIIIA was present in the intercellular space in well-formed cytotrophoblastic shells, while the cytotrophoblastic shells and Nitabuch's layers in this patient's implantation tissue were poorly-formed. Furthermore, XIIIA was not detected around them. It is suggested that when the maternal plasma activity of factor XIII is low, the concentration of XIIIA at the placental bed is also low, leading to the insufficient formation of cytotrophoblastic shell and therefore an increased probability of miscarriage in patients with congenital factor XIII deficiency.

Solid-state ¹³C two-dimensional (2D) exchange NMR has been applied to investigate dynamic alternation among three forms of hydrogen bonds in the poly(acrylic acid)/poly(ethylene oxide) (PAA/PEO) complex. The ¹³C CPMAS NMR spectrum of the signal for the PAA carboxyl carbon in the complex is split into three peaks corresponding to three forms of hydrogen bonds, namely, the complex form, the dimeric form, and the free form. These three peaks coalesce into a single peak at temperatures above 346 K, showing that dynamic alternation of hydrogen bonds occurs in the complex. 2D exchange NMR spectra directly show that the dissociation−association of hydrogen bonds occurs at temperatures above 329 K, which is 7 deg higher than the glass transition temperature (Tg). The exchange rates are on the order of Hz and have a wide distribution. At the same time, it was found that PAA conformational transitions occur at a rate comparable to that of the hydrogen bond alternation. On the other hand, the PEO motion shows no apparent correlation to the hydrogen-bonding dynamics. It is concluded that the hydrogen-bonding dynamics is coupled to the segmental motion of PAA in the complex.

The aim of this research was to study the effect of exogenous interleukin (IL)-8, IL-1 and IL-8 + IL-1 on uterine contractions in rabbits. Four equal groups of non-pregnant rabbits (n = 24) were investigated using either placebo or experimental drugs in the form of vaginal suppositories. The suppositories contained human recombinant IL-8 (200 ng), IL-1 (200 ng), IL-8 (200 ng) + IL-1 (200 ng) or vehicle Witepsol base, 500 microliters). Subsequently, the plasma concentration of prostaglandin (PG) E2 was estimated 3 h after the last dose of treatment. Neutrophil infiltration in the endometrial tissue was studied with anti-rabbit RT2 staining. Suppositories with IL-1 and IL-8 + IL-1 produced contractile responses with increased frequency (P < 0.003, P < 0.0005) and amplitude (P < 0.0001) in vivo, compared with vehicle. IL-1 and IL-8 + IL-1 also caused similar contractile effects with increased frequency (P < 0.01, P < 0.0007) and amplitude (P < 0.0001) in an in-vitro experiment than vehicle. The frequency and amplitude of uterine contractions were more significant with IL-8 + IL-1 than that of IL-1, both in vivo (P < 0.002, P < 0.05) and in vitro (P < 0.005, P < 0.01). IL-8 did not induce any contractions. Prostaglandin concentration was increased approximately 8-fold with IL-8 + IL-1 (P < 0.0001) and 2.5-fold with IL-1 treatment (P < 0.0001). Neutrophil numbers were significantly increased with IL-8 + IL-1 > IL-8 > IL-1 (P < 0.002, P < 0.0003 and P < 0.008) compared with vehicle. Our data suggest that IL-8 stimulates IL-1-induced uterine contractions through PGE2 production and could be an important process during labour and delivery.

To study the intrauterine defensive role of fetal urine, we focused urinary trypsin inhibitor (UTI) from fetal urine. Urinary trypsin inhibitor (UTI) is a glycoprotein secreted with fetal urine. Until now, its real function has been unknown. The expression of IL-8 induced by LPS were completely abolished in the presence of physiological concentration of UTI in various cells. LPS or IL-8 in the form of vaginal suppositories induced cervical ripening in rabbits. UTI applied after LPS or IL-8 could successfully inhibit the changes induced by IL-8 or LPS in the rabbit cervix. UTI or fetal urine inhibits the increase of intracellular Ca^2^+ of uterine muscle cells induced by LPS. Possibly, this regulatory effect of intracellular Ca^2^+ is involved in preventing uterine muscle contraction. UTI or fetal urine suppresses premature cervical ripening and prevents uterine muscle contraction. We conclude that UTI of fetal urine is important role in maintaining pregnancy.

It is well known that maternal fibrinogen (Fg) and subunit A of factor XIII (XIIIA) are essential for maintaining early pregnancy. We reported that they were essential from 5 weeks' gestation and were present immunohistochemically at the implantation site. Now we studied the precise localization of Fg, fibronectin (Fn), XIIIA, and subunit S of factor XIII (XIIIS) associated with the development of extravillous cytotrophoblast (EV-CTr) in the human implantation tissues and placenta. Cytotrophoblast cells were confirmed by cytokeratin immunolabeling. The results showed that in the region around the early placental bed, Fg and Fn were present at Nitabuch's layer, and XIIIA was present at the extracellular space of EV-CTrs forming the cytotrophoblastic shell adjacent to Nitabuch's layer. In the decidual stroma near Nibabuch's layer, where interstitial EV-CTrs were infiltrating, Fn was present. However, any obvious localization of Fg, XIIIA and XIIIS could not be detected. In the decidual stroma far from Nitabuch's layer, where interstitial EV-CTrs did not infiltrate, numerous XIIIA positive macrophages were detected. However, any obvious localization of Fg, Fn, plasma and platelet XIIIA, and XIIIS could not be detected. Discussion of the results leads to a hypothesis that maternal Fg, maternal XIIIA and Fn present at the site around the cytotrophoblastic shell are associated with the early placental adhesion. Also, Fn is present at the decidual stroma where interstitial EV-CTrs are infiltrating, so it may be oncofetal Fn and associated with trophoblastic outgrowth and migration. XIIIA positive macrophages and the interstitial EV-CTrs cannot be present together at the decidual stroma. However, XIIIA positive macrophages may play a role in assisting the tetomaternal adhesion.

To investigate the role of phosphatidylserine (PS) derived from the activated platelets in placental circulation, we established an artificial PS-induced intrauterine growth restriction (IUGR) model in mice and examined whether annexin V, a PS-binding anticoagulant protein, could prevent the development of IUGR in the animal experiments. The ICR mice were injected in the tail vein on days 8, 11 and 14 of pregnancy with filtered PS/phosphatidylcholine (PC) microvesicles (<0.3μm in maximal diameter). The microvesicles have a procoagulant activity which was inhibited by annexin V in a dose-dependent fashion. The mice were killed on day 18 of pregnancy and the placental and fetal weights were measured. The placental tissue specimens were examined microscopically. PS/PC vesicles induced significant reductions in fetal weights compared with PC vesicles alone. The placental tissue revealed severe congestion with fibrin depositions although the lung and kidney tissue specimens showed minimal histological changes. PS/PC microvesicles with recombinant annexin V showed no significant reductions in fetal weights in mice with PS/PC vesicles alone. These results suggest that PS from the activated platelets induces IUGR by enhancing the coagulation cascade in the placental circulation and that annexin V from the villous trophoblast prevents the development of IUGR.

Hyaluronic acid (HA) stimulates the synthesis of interleukin (IL) 8, while dehydroepiandrosterone sulphate (DHEA-S) induces the expression of IL-8 and its receptor in the human cervical fibroblast. This has led us to investigate the effect of DHEA-S on HA-induced cervical ripening. Experiments were performed in pregnant rabbits using vaginal suppositories containing 1 mg HA, 30 mg DHEA-S, 30 mg DHEA-S + 0.1 mg HA, 30 mg DHEA-S + 1 mg HA, and 500 microl Witepsol-50 base (control). The effects were evaluated by measuring collagenase, gelatinase and elastase activities, water content, neutrophil infiltration, relative collagen concentration and histological assessment. The activities of collagenase, gelatinase and elastase were significantly increased in rabbits treated with DHEA-S + 1 mg HA compared with rabbits treated with DHEA-S + 0.1 mg HA (P < 0.009, P < 0.001, P < 0.009 respectively). Water content was markedly increased in rabbits treated with DHEA-S + 1 mg HA compared with DHEA-S + 0.1 mg HA treatment (P < 0.05). Neutrophil infiltration was markedly increased, while relative collagen concentration was significantly decreased with DHEA-S + 1 mg HA compared with the DHEA-S + 0.1 mg HA approach (P < 0.001, P < 0.002). The histology of cervices treated with DHEA-S + 1 mg HA showed the density of collagen to be markedly decreased, and collagen fibres irregularly separated. Increased vascularity with massive dilatation of blood vessels was also observed in these rabbits. We conclude that DHEA-S upregulates the HA-induced cervical ripening process.

From high-resolution solid-state13C NMR spectra for the enol forms of some diketones, an average chemical shift of the carbonyl and enol carbons in dimedone is found to move remarkably downfield compared with the corresponding shift in DMSO-d6, which is ascribed to a strong intermolecular hydrogen bond in the solid state.

Throughout Japan a total of 543 cases of vitamin K deficiency occurring in infants over 2 weeks of age were reported from January 1981 to June 1985. Of these cases, 427 showed no obvious reasons for vitamin K deficiency; this sort of case is known as “idiopathic vitamin K deficiency in infancy”. Another 57 cases had bleeding episodes due to vitamin K deficiency associated with obvious hepatobiliary lesions, chronic diarrhoea, long-term antibiotic therapy, etc; this sort is called “secondary vitamin K deficiency in infacy”. The third group, consisting of 59 cases, was made up of the socalled “near miss” type, in which a haemorrhagic tendency, without any obvious clinical haemorrhage, was discovered by Normotest, at the time of mass screening in most cases. In the idiopathic group, 269 cases (63.0%) developed bleeding episodes between the 1st and 2nd months of age, and 387 cases (90.0%) were entirely breast-fed. Intracranial baemorrhage was observed in 353 cases (82.7%) of this group. Moreover, slight elevation of serum transaminase and direct type bilirubin levels were observed in the idiopathic group. Liver dysfunction of unknown origin may play some role in the onset of vitamin K deficiency in infancy.

The effect of dibutyryl cAMP on dystrophin mRNA expression was investigated in C2C12 myogenic cells by a semi-quantitative RT-PCR method. The dystrophin mRNA level was enhanced by the addition of low concentrations of dibutyryl cAMP for 16h in the growth medium. The relative amounts of dystrophin/G3PDH mRNA at 10−4M dibutyryl cAMP were about 2.6-fold higher compared to control. We examined MLCla mRNA expression in order to know whether the induction of dystrophin mRNA is due to the facilitation of myogenic differentiation by dibutyryl cAMP. MLCla mRNA expression at 10−4M dibutyryl cAMP was similar to that of control. Thus we conclude that the dibutyryl cAMP enhancement of dystrophin mRNA expression is not due to a secondary event involving the formation of differentiated myotubes. The induction of CREB mRNA by dibutyryl cAMP suggests that the up-regulation of dystrophin mRNA expression might occur via transcriptional activation.

The effect of lithium on thyroid function was studied in 18 neuroleptic-treated male chronic schizophrenic in-patients. Lithium carbonate was administered for 8 weeks at a dosage giving a mean serum level of 0,79 mmol/l. Blood was obtained just before and after 8 weeks of lithium administration to determine the serum free thyroxine (free T4) levels, free triiodothyronine (free T3) levels and thyrotropin (TSH) levels. Overall, free T4and TSH levels significantly increased whereas free T3levels did not change. Two (11 ) patients had abnormally increased free T, levels and abnormally decreased free T, levels after 8 weeks of lithium administration. These findings suggest that lithium may inhibit the peripheral conversion of free T4to free T3in some susceptible patients.

Single-chain urokinase-type plasminogen activator (pro-uPA) is bound to a specific surface receptor on ovarian cancer HOC-I cells that is incompletely saturated. Saturation of uncovered receptors by uPA polypeptides with intact amino-terminal fragment (ATF) derived from pro-uPA by limited proteolysis (human leucocyte elastase [HLE] or V8 protease) has been studied. HOC-I cells preferentially invaded reconstituted basement membranes in a time- and plasminogen-dependent manner. This process was inhibitable by preincubation with uPA polypeptides in the medium at levels which suggested that complete saturation of cell surface uPA receptors occurred. This result indicates that occupation of uPA receptors by enzymatically inactive uPA fragments or prevention of rebinding of pro-uPA synthesised by tumour cells to the receptors specifically reduces the invasion of the tumour cells through basement membranes in vitro.

Proton spin–lattice relaxation times (T1) were measured on a protein Streptomycessubtilisin inhibitor, in the solid state, in the presence and the absence of adsorbed 2H2O in the temperature range between −170 and 100 °C at 30 and 60 MHz. In both samples, a common relaxation process is attributed to the methyl group rotations of amino acid side chains which show minimum values of T1of 0.11 s at −105 °C at 30 MHz and 0.23 s at −80 °C at 60 MHz. In the presence of adsorbed 2H2O, another, effective relaxation process was found above 0°C, showing a minimum value of T1of 0.12 s at about 80 °C at 60 MHz. The latter relaxation process was attributed to internal motions of exposed peptide segments, a large part of which is located in the crucial enzyme-binding region of the inhibitor.

Inter-alpha-trypsin inhibitor (ITI), a human serum protease inhibitor of molecular mass 240 kDa which may release physiological derivatives, has been shown to interact with hyaluronic acid (HA), resulting in pericellular matrix stabilization (Chen, L., Mao, S.J.T., McLean, L. R., Powers, R. W., and Larsen, W. J. (1994) J. Biol. Chem. 269, 28282-28287). The purpose of this study is to determine whether ITI binding to tumor cell surface is mediated by urinary trypsin inhibitor (UTI)-receptor or cell-associated hyaluronic acid (HA). We demonstrated specific complex formation of the heavy (H) chains of ITI with HA. Binding of the H-chains of ITI to immobilized HA was detected and quantified using colorimetric immunoassays. Binding was time-, temperature-, and concentration-dependent. However, UTI and HI-8 (the carboxyl terminus of UTI) failed to bind to immobilized HA. ITI bound to HA remained functional protease inhibiting activity. After incubation of SMT-cc1 cells with purified biotinylated ITI, biotinylated ITI is bound to the cells, dissociated, and gives rise to the H-chains and UTI on the cell surface. The cell surface receptor-bound UTI derived from ITI may be the result of the limited proteolysis on the cell surface. In the cells treated with hyaluronidase, bound H-chains disappeared from the surface of the cells, while most of the cell surface ITI derivatives was present in deglycosylated UTI (28 kDa). It is suggested that the binding of ITI to the cell surface is mediated by HA on the cells. This was confirmed by the fact that the hyaluronidase-treated cells can abolish the ITI binding. The cell surface UTI formation was inhibited by diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, and eglin C, suggesting that elastase-like enzyme(s) may be responsible for the UTI formation. Preincubation of the cells with UTI did not decrease in exogenously added ITI on the cell surface. A model for cell surface UTI formation is proposed in which ITI binding to cells from serum used for the culture is followed by the limited proteolysis by trace amounts of active serine proteases, to form cell-surface receptor-bound UTI and the H-chains intercalated into cell surface HA. This process is subject to regulation of cell-associated UTI and of stabilization of pericellular matrix.