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Cocrystallization is a technique for improving the physical properties of active pharmaceutical ingredients. However, cocrystals can transform into more stable polymorphs as well as dissociate to original materials. Therefore, an analytical technique is required to determine the polymorphic transformation quickly and accurately in tablets. The purpose of this study is to develop a method to monitor cocrystal polymorphs in model tablets using transmission low-frequency Raman spectroscopy. The tablets, consisting of only metastable polymorphs of caffeine-glutaric acid cocrystals, were stored under various relative humidity levels. The composition of the cocrystal polymorphs were calculated from a calibration curve relating the actual composition to the predicted values calculated by partial least squares regression processing of low-frequency Raman spectra. The metastable form gradually converted to a stable form, and polymorphic phase transformation occurred with increasing relative humidity. Ninety-six percent of the metastable form converted into a stable form stored at 25 °C after 3 h at 95% RH. In conclusion, transmission low-frequency Raman spectroscopy can be used to quantitatively monitor cocrystal polymorphs. This technique is one of the candidate techniques to quantifiably evaluate the physico-chemical stability of cocrystal polymorphs in tablets., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

α-Crystallin, comprising 40-50 subunits of αA- and αB-subunits, is a long-lived major soluble chaperone protein in lens. During aging, α-crystallin forms aggregates of high molecular weight (HMW) protein and eventually becomes water-insoluble (WI). Isomerization of Asp in α-crystallin has been proposed as a trigger of protein aggregation, ultimately leading to cataract formation. Here, we have investigated the relationship between protein aggregation and Asp isomerization of αA-crystallin by a series of analyses of the soluble α-crystallin, HMW and WI fractions from human lens samples of different ages (10-76 years). Analytical ultracentrifugation showed that the HMW fraction had a peak sedimentation coefficient of 40 S and a wide distribution of values (10-450 S) for lens of all ages, whereas the α-crystallin had a much smaller peak sedimentation coefficient (10-20 S) and was less heterogeneous, regardless of lens age. Measurement of the ratio of isomers (Lα-, Lβ-, Dα-, Dβ-) at Asp58, Asp91/92 and Asp151 in αA-crystallin by liquid chromatography-mass spectrometry showed that the proportion of isomers at all three sites increased in order of aggregation level (α-crystallin < HMW < WI fractions). Among the abnormal isomers of Asp58 and Asp151, Dβ-isomers were predominant with a very few exceptions. Notably, the chaperone activity of HMW protein was minimal for lens of all ages, whereas that of α-crystallin decreased with increasing lens age. Thus, abnormal aggregation caused by Asp isomerization might contribute to the loss of chaperone activity of α-crystallin in aged human lens., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Collagen is the most abundant protein in the animal kingdom and has a unique triple-helical structure. It not only provides mechanical strength to tissues, but also performs specific biological functions as a multifaceted signaling molecule. Animal-derived collagen is therefore widely used as a biocompatible material in vitro and in vivo. In this study, we developed a novel peptide-based material that mimicked both the polymeric properties and a selected biological function of native collagen. This material was prepared by end-to-end multiple disulfide cross-linking of chemically synthesized triple-helical peptides. The peptide polymer showed a gel-forming property, and receptor-specific cell binding was observed in vitro by incorporating a peptide harboring an integrin α2β1-binding sequence. Furthermore, cell signaling activity and biodegradability were tunable according to the polymer contents. The results demonstrated the potential of this material as a designer collagen. STATEMENT OF SIGNIFICANCE: Collagen is a useful biomaterial with the gel-forming property. It also exhibits various biological activities through the interaction of specific amino acid sequences displayed on the triple helix with functional biomacromolecules. Here we report a novel synthetic material, artificial collagen, by end-to-end cross-linking of chemically synthesized collagen-like triple-helical peptides. The material allows independent regulation of polymer properties, i.e. gel stiffness, and sequence-specific bioactivities by altering peptide compositions. This material can also be variously shaped, for example, thin films with high transparency. In addition, it has low inflamatogenic properties and tunable biodegradability in vivo., (Copyright © 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Background: To examine whether combined use of 123 I-FP-CIT dopamine transporter single photon emission computed tomography (DAT-SPECT) and 123 I-MIBG myocardial scintigraphy (MIBG) is superior to either modality alone for diagnosing Parkinson's disease (PD)., Methods: Patients with probable PD (n=120) who underwent both DAT-SPECT and MIBG myocardial scintigraphy within short intervals were enrolled. Specific binding ratio (SBR) of DAT-SPECT images and heart-to-mediastinum (H/M) ratio of MIBG images were used as quantitative measures. We classified patients into 4 groups based on SBR value and H/M ratio, or into two groups based on the striatal asymmetry index (SAI) of DAT-SPECT, and examined the clinical features of each group. We also investigated the characteristics of SWEDDs (scans without evidence of dopaminergic deficits) patients. Finally, we calculated the sensitivity and specificity of each method and the combined method., Results: SBR value was significantly correlated with both early and delayed H/M ratio values. Motor complications and hallucinations were observed at high frequency in the group with both lower SBR and H/M ratio, and hallucinations appeared in the group with larger SAI. SWEDDs were observed 8.3% of patients. The sensitivity and specificity of diagnosing PD were 91.7% and 15.0% by SBR of DAT-SPECT, 78.3% and 90.0% by H/M ratio of MIBG uptake, and 74.2% and 95.0% by the combined modalities, respectively., Conclusions: Combined use of DAT-SPECT and MIBG myocardial scintigraphy increases the specificity of PD diagnosis, and is helpful for understanding the clinical features or predicting complications., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Bilious pleural effusion is an extremely rare condition associated with liver diseases, subphrenic or subhepatic abscess formation, biliary peritonitis, and invasive procedures (i.e., percutaneous biliary drainage or liver biopsy). The current diagnostic test is based on the measurement of the ratio of pleural total bilirubin to serum total bilirubin, which is greater than 1 in patients with bilious pleural effusion. Given the low incidence of bilious pleural effusion, the precise diagnostic yield of this ratio based test has not been evaluated., Methods: We retrospectively reviewed the medical records of our institution and searched the PubMed database for reports of bilious pleural effusion., Results: We identified a total of 12 cases of bilious pleural effusion (9 from 8 Pubmed reports and 3 from our institutional records). The factors causing this condition were broadly classified into three categories based on the pathophysiology: 1) liver diseases (echinococcosis, tuberculosis and amebiasis); 2) subhepatic/subphrenic abscess or biliary peritonitis, with or without biliary tract obstruction; and 3) iatrogenic disease after percutaneous biliary drainage and/or liver biopsy. The sensitivity of detection was 76.9% when the ratio of pleural total bilirubin to serum total bilirubin was greater than 1. The sensitivity increased to 100% when a combination test including pleural glycoholic acid was adopted., Conclusions: This study demonstrates the high diagnostic yield for bilious pleural effusion using a combination of two test criteria; a ratio of pleural total bilirubin to serum total bilirubin greater than 1 and the presence of pleural glycoholic acid., (Copyright © 2016 The Japanese Respiratory Society. Published by Elsevier B.V. All rights reserved.)

Purpose: Congestive heart failure (CHF) is one of the risk factors for deep vein thrombosis (DVT) according to the Japanese guidelines for DVT treatment and prevention. The purpose of this study is to estimate the frequency of DVT among hospitalized CHF patients, since there have been only limited DVT data in Japanese CHF patients., Methods: Patients enrolled in the study were with risk factors for DVT listed in the guidelines as well as with acute exacerbation of CHF, bed rest for at least 4 days, and aged 60 or above. Patients treated by physical prophylaxis or anti-platelet medication were included, while patients treated by any anticoagulant medicines were excluded. Patients with surgery or injury within 3 months before the hospitalization or diagnosed clinically or with obvious past history as having DVT at hospitalization were excluded. The presence of DVT in the eligible patients was determined by ultrasonography and the images were evaluated by an independent central evaluation committee., Results: Forty-four patients were enrolled in the study including 19 males and 25 females. The mean age was 79.0±10.6 years, and the mean duration of bed rest was 8.9±3.2 days. Out of these 44 patients, DVT was detected in 15 (34%) patients. Eight patients were on treatment with physical prophylaxis but DVT was still detected in two patients. Furthermore, 12 out of the rest of the patients were treated by anti-platelet agents and were still with DVT in 3 patients., Conclusion: When evaluated ultrasonographically, the frequency of DVT in hospitalized non-surgical Japanese patients with CHF was approximately 35%. DVT occurred in 25% of patients treated by physical prophylaxis or anti-platelet agents. The results suggest that Japanese hospitalized patients with CHF have a high risk of DVT and thus can be recognized to have potential benefit by preventing and treating DVT according to the guidelines., (Copyright © 2014 Japanese College of Cardiology. Published by Elsevier Ltd. All rights reserved.)

For the longevity of total knee joint prostheses, we have developed an artificial lubricant using polyethylene glycol (PEG) for the prevention of wear of ultra-high-molecular-weight polyethylene (UHMWPE). In the present study, the lubricative function of this PEG lubricant was evaluated by a wear test using Co-Cr alloy and UHMWPE counter surface samples. As a result, human synovial fluid including the PEG lubricant showed good result regarding the wear volume and a worn surface of UHMWPE. Considering its lubrication mechanism, it is suspected that interaction between the PEG molecules and the proteins in synovial fluid was involved. Since PE molecules are also organic compounds having a hydroxyl group at one or both ends, the albumin and PEG molecule complex would have bound more strongly to the metal oxide surface and UHMWPE surfaces might enhance and stabilize the lubricating film between the contact surfaces under the boundary lubrication. This study suggests that PEG lubricant as an intra-articular viscous supplement has the potential to prevent wear of UHMWPE by mixing with synovial fluid and to contribute to the longevity of knee joint prostheses., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Objective: The characteristic pathological muscular findings of polymyositis (PM) and dermatomyositis (DM) have been shown to reflect their different pathogeneses. Here, we characterized the muscle biopsy findings of PM and DM patients with or without malignancy., Methods: We evaluated the muscle biopsy findings of 215 consecutive PM and DM patients admitted to our hospital between 1970 and 2009. Pathology of the lesion biopsy sections was classified into 3 types: endomysial infiltration-type, perivascular infiltration-type, and rare-infiltrative-type., Results: There was no difference between the muscle pathology of PM patients with and without malignancy. However, the incidence of rare-infiltrative type muscle pathology in DM patients with malignancy was significantly higher than in those without such tumors (p=0.0345)., Conclusion: The incidence of rare-infiltrative type muscle pathology may be a predictive marker of DM with malignancy., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: The differences in the small intestinal toxicity of low-dose aspirin based on the type of aspirin used remains unclear. The purpose of this study was to evaluate the differences in the small bowel mucosal injury between buffered and enteric-coated aspirin users by capsule endoscopy., Methods: We retrospectively reviewed the findings in chronic low-dose aspirin users (>3 months) who underwent capsule endoscopy for the investigation of obscure gastrointestinal bleeding. The patients were classified into two groups based on the type of low-dose aspirin that they had been prescribed (enteric-coated aspirin group or buffered aspirin group), and evaluated the numbers of small bowel lesions and the Lewis score., Results: Capsule-endoscopic findings of a total of 70 patients taking low-dose aspirin were reviewed. Significant differences in the number of erosions and ulcers were observed between the buffered and enteric-coated aspirin groups (P=0.017 and P=0.037, respectively). The median Lewis score for the small bowel mucosal inflammatory change was significantly higher in the enteric-coated aspirin group than in the buffered aspirin group (P=0.035)., Conclusions: The results of this study suggested that enteric-coated aspirin might be more injurious to the small bowel mucosa than buffered aspirin., (Copyright © 2012 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Background and Aims: In the present study, we describe a newly developed microchip-based analytical system to evaluate white thrombus formation (WTF). Efficacies of various antithrombotic agents were compared under different flow conditions., Methods: Whole blood containing corn trypsin inhibitor was perfused over a microchip coated with collagen and tissue thromboplastin at the lower and higher shear rates of 240 and 600 s(-1) , and WTF process inside the microchip was quantified by monitoring a flow pressure. Parameters of T(10) (time to 10 kPa), T(10-80) (time from 10 to 80 kPa) and OT (occlusion time; time to 80 kPa) were used to evaluate the onset and the growth rate of WTF, and the capillary occlusion, respectively., Results: After perfusion was started, white thrombus composed of activated platelets and fibrin was formed on the coated surface. Thrombus gradually increased in size and eventually occluded the capillary. Among anticoagulants, heparin (0.5-1.0 U mL(-1)) potently prolonged T(10) at both shear rates, whereas low molecular weight heparin (1.0-2.0 IU mL(-1)) inhibited the growth of WTF at the lower shear rate. Among antiplatelet agents, abciximab (1-2 μg mL(-1)) significantly reduced the size and number of thrombi, which was additively enhanced in the presence of heparin (0.5 U mL(-1) ). OS-1 (specific GPIbα-antagonist) prevented the complete capillary occlusion., Conclusion: The novel monitoring system of WTF may be useful in preclinical and clinical evaluations of different types of antithrombotic strategies, and their effects in combination., (© 2011 International Society on Thrombosis and Haemostasis.)

Vicenin-3 was synthesized from naringenin via a short five-step reaction, which included two regioselective direct C-glycosylations with d-glucose and d-xylose (yields: 22% and 30%, respectively) as the key reactions for a total yield of 4.4%. Vicenin-1 was also synthesized from phloroacetophenone via a 10-step reaction, including the same glycosylation described above, for a total yield of 2.7% with a vicenin-3 yield of 1.7%.

Naofen has recently been identified from the rat brain/spinal cord cDNA library as a substance reactive against an anti-shigatoxin (Stx)-2 antibody. Naofen mRNA is composed of 4620 nucleotides and encodes 1170 amino acids. Naofen contains four WD-repeat domains in its N-terminus and is ubiquitously distributed in many tissues of the rat. Tumor necrosis factor (TNF)-alpha enhanced the expression of naofen mRNA in HEK293 cells in a dose-dependent manner. Furthermore, naofen siRNA, which predominantly knocked down the expression of naofen mRNA, significantly reduced both TNF-alpha-induced caspase-3 activation and apoptosis in HEK293 cells. Overexpression of naofen in HEK293 cells (FLAG-NF) spontaneously induced caspase -3 activation and apoptosis, and showed extremely high susceptibility to TNF-alpha-induced apoptosis. These results indicated that naofen may function as a novel modulator activating caspase-3, and promoting TNF-alpha-stimulated apoptosis., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Collagen-binding proteins (CBPs) play important roles in various physiological events. Some CBPs are regarded as targets for drug development; for example, platelet glycoprotein VI (GPVI) and heat shock protein 47 (HSP47) are promising targets for the development of novel antiplatelet and antifibrotic drugs, respectively. However, no systematic screening method to search compounds that inhibit collagen-CBP interactions have been developed, and only a few CBP inhibitors have been reported to date. In this study, a facile turbidimetric multiwell plate assay was developed to evaluate inhibitors of CBPs. The assay is based on the finding that CBPs retard spontaneous collagen fibril formation in vitro and that fibril formation is restored in the presence of compounds that interfere with the collagen-CBP interactions. Using the same platform, the assay was performed in various combinations of fibril-forming collagen types and CBPs. This homogeneous assay is simple, convenient, and suitable as an automated high-throughput screening system.

Histidine-rich glycoprotein (HRG) promoted the adhesion and morphological changes of human T-cell line MOLT-4 in a Con A-dependent manner. This morphological change-promoting activity was specific for HRG and the Arg23-Lys66 glycopeptide from human HRG. The carbohydrate chain at Asn45 was essential for this activity. The morphological changes of MOLT-4 cells caused by HRG and Con A (HRG/Con A) were not inhibited by phosphatidylinositol 3-kinase inhibitor, wortmannin or LY294002, while the changes by Con A alone were completely inhibited by these reagents, suggesting that HRG/Con A cooperate to activate leukocytes via a signaling pathway distinct from that by Con A alone. The morphological changes by Con A were associated with pseudopodia like structure. On the other hand, the morphological changes caused by HRG/Con A were associated not only with pseudopodia like structure but also with an increase of the F-actin-rich surface protrusions. Wortmannin inhibited only the formation of pseudopodia like structure.

Although posterior reversible encephalopathy syndrome (PRES) is caused by various conditions, there have been no reports on PRES associated with bronchial asthma. We report a case with PRES during the treatment for severe asthmatic attack. A 4-year-old girl was treated for asthmatic attack with steroids. From the 10th hospital day, hypertension, pulmonary edema, and cardiomegaly were observed. In spite of treatment with furosemide, she became lethargic and had a generalized convulsion on the 23rd hospital day. CT showed low density areas in the bilateral occipital white matter and MRI on the 28th hospital day demonstrated high intensity areas in the same regions on T2-weighted and FLAIR images. After discontinuation of corticosteroid and further antihypertensive therapy, her consciousness improved. MRI on the 67th hospital day had no abnormalities and no neurological sequelae were seen at 2 years after the event. We should be aware that PRES is a rare but important adverse event related to steroid therapy, because hypertension and water retention are major adverse effects of steroids.

Background: Thrombin is a primary inducer of thrombus formation by activations of coagulation cascade and platelet aggregation. Hitherto, several types of thrombin inhibitors have been developed for therapeutic purpose., Objectives: We prepared modified thrombin (M-thrombin) and modified anhydrothrombin (M-anhydrothrombin) by chemical modification of carboxyl groups of thrombin and anhydrothrombin, respectively, to present a new strategy for a potent antiplatelet-anticoagulant agent and new tools for investigation of thrombin functions., Results: M-anhydrothrombin retained high affinity for factor VIII (FVIII), but demonstrated lower affinity than anhydrothrombin for fibrinogen and factor V (FV). Both M-anhydrothrombin and anhydrothrombin prolonged activated partial thromboplastin time (APTT) without affecting prothrombin time, and M-anhydrothrombin prolonged APTT much more than anhydrothrombin. M-anhydrothrombin also retained affinity for the recombinant extracellular domain peptide of protease-activated receptor 1 (PAR1). M-thrombin exhibited marginal clotting activity (4% of thrombin), but induced platelet aggregation in platelet-rich plasma without forming a fibrin clot, which was completely suppressed by anti-PAR1 antibody (ATAP2) and by M-anhydrothrombin, but not by anhydrothrombin. These results indicate that M-thrombin induced platelet aggregation through the activation of PAR1, and M-anhydrothrombin inhibited this process completely. In contrast, neither M-anhydrothrombin nor anhydrothrombin apparently inhibited thrombin-induced platelet aggregation. Only in the presence of the Gly-Pro-Arg-Pro (GPRP) peptide that inhibits polymerization of fibrin, M-anhydrothrombin completely inhibited thrombin-induced platelet aggregation., Conclusion: M-thrombin is PAR1-specific and M-anhydrothrombin is FVIII- and PAR1-specific derivatives, and thereby, are new tools as specific agonist and antagonist, respectively, of PAR1. Furthermore, M-anhydrothrombin may be an attractive model for development of a potent anticoagulant-antiplatelet agent.

Pigment epithelium-derived factor (PEDF) is the most potent endogenous inhibitor of angiogenesis in age-related macular degeneration and tumors. However, the molecular mechanism of the anti-angiogenic activity of PEDF is poorly understood. PEDF interacts with the extracellular matrix (ECM) in vitro. Here, we investigated the possible involvement of the motif for ECM interaction in the anti-angiogenic activity of PEDF. The growth rates of HeLa cells in culture were not affected by transfection of PEDF, indicating that PEDF did not suppress tumor cell growth directly. In tumor xenografts, the overexpression of wild-type PEDF significantly suppressed tumor growth, whereas a mutant of the collagen I-binding site of PEDF (Col-mut PEDF) did not inhibit tumor growth. A mutant of the heparin-binding site of PEDF (Hep-mut PEDF) suppressed tumor growth. Histological analysis showed that the density and area of microvasculatures in either PEDF or Hep-mut PEDF were suppressed when compared with those in either vector or Col-mut PEDF. Our data indicate that PEDF inhibits tumor growth via its anti-angiogenic activity, and the collagen I-binding motif of PEDF is involved in the biological activity.

Histidine-rich glycoprotein (HRG) is a serum protein belonging to the cystatin superfamily. HRG may play a regulatory role in hemostasis and innate immunity. However, this role is uncertain because of a lack of rigorous testing in an animal model. We generated mice lacking the translation start point of exon 1 of the Hrg gene, effectively resulting in a null mutation (Hrg-/-). The mice were viable and fertile but had no HRG in their blood. Antithrombin activity in the plasma of Hrg-/- mice was higher than in the plasma of heterozygous Hrg+/- or wild-type Hrg+/+ mice. The prothrombin time was shorter in Hrg-/- mice than in Hrg+/- and Hrg+/+ mice. Bleeding time after tail tip amputation in Hrg-/- mice was shorter than in Hrg+/+ mice. The spontaneous fibrinolytic activity in clotted blood of Hrg-/- mice was higher than in Hrg+/+ mice. These findings suggest that HRG plays a role as both an anticoagulant and an antifibrinolytic modifier, and may regulate platelet function in vivo.

We have undertaken a large-scale microarray gene expression analysis using cDNAs corresponding to 21,000 Xenopus laevis ESTs. mRNAs from 37 samples, including embryos and adult organs, were profiled. Cluster analysis of embryos of different stages was carried out and revealed expected affinities between gastrulae and neurulae, as well as between advanced neurulae and tadpoles, while egg and feeding larvae were clearly separated. Cluster analysis of adult organs showed some unexpected tissue-relatedness, e.g. kidney is more related to endodermal than to mesodermal tissues and the brain is separated from other neuroectodermal derivatives. Cluster analysis of genes revealed major phases of co-ordinate gene expression between egg and adult stages. During the maternal-early embryonic phase, genes maintaining a rapidly dividing cell state are predominantly expressed (cell cycle regulators, chromatin proteins). Genes involved in protein biosynthesis are progressively induced from mid-embryogenesis onwards. The larval-adult phase is characterised by expression of genes involved in metabolism and terminal differentiation. Thirteen potential synexpression groups were identified, which encompass components of diverse molecular processes or supra-molecular structures, including chromatin, RNA processing and nucleolar function, cell cycle, respiratory chain/Krebs cycle, protein biosynthesis, endoplasmic reticulum, vesicle transport, synaptic vesicle, microtubule, intermediate filament, epithelial proteins and collagen. Data filtering identified genes with potential stage-, region- and organ-specific expression. The dataset was assembled in the iChip microarray database, , which allows user-defined queries. The study provides insights into the higher order of vertebrate gene expression, identifies synexpression groups and marker genes, and makes predictions for the biological role of numerous uncharacterized genes.

We analyzed mutations of 7 vitamin K-dependent protein and cytochrome P450 2C9 genes in 45 patients and investigated whether any contribute to the large interpatient variability in the warfarin dose-effect relationship. Total clearance and daily dose, INR and INR/Cp, were used as pharmacokinetic and pharmacodynamic indexes, respectively. Patients were grouped by genotype based on a single polymorphism and combinations of polymorphisms. Among the 30 sequence variants identified, CYP2C9*3, 165Thr-->Met of the factor II gene, -402G-->A, (37-bp repeat)n, and -746T-->C of the factor VII gene, and (CAA repeat)n of the gamma-glutamyl carboxylase gene were selected as candidate polymorphisms. As the analysis of single polymorphisms implied, the highest INR/Cp mean values and the lowest warfarin maintenance doses were observed in patients homozygous for the 165Met, -402G, (37-bp repeat)6 and -746T alleles. Multiple regression analysis revealed that warfarin sensitivity was independently associated with -402G-->A, (CAA repeat)n, CYP2C9*3, and 165Thr-->Met, which accounted for 50% of variance. These results suggest that part of the considerable interpatient variation is attributable to genetic variation, and the combined genotyping of CYP2C9 and certain vitamin K-dependent protein genes is useful for predicting anticoagulant responses.

Previously we showed that two antithrombin mutants were degraded through an endoplasmic reticulum (ER)-associated degradation (ERAD) pathway [F. Tokunaga et al., FEBS Lett. 412 (1997) 65]. Here, we examined the combined effects of inhibitors of glycosidases, protein synthesis, proteasome, and tyrosine phosphatase on ERAD of a Glu313-deleted (DeltaGlu) mutant of antithrombin. We found that kifunensine, an ER mannosidase I inhibitor, suppressed ERAD, indicating that specific mannose trimming plays a critical role. Cycloheximide and puromycin, inhibitors of protein synthesis, also suppressed ERAD, the effects being cancelled by pretreatment with castanospermine. In contrast, kifunensine suppressed ERAD even in castanospermine-treated cells, suggesting that suppression of ERAD does not always require the binding of lectin-like ER chaperones-like calnexin and/or calreticulin. These results indicate that, besides proteasome inhibitors, inhibitors of ER mannosidase I and protein synthesis suppress ERAD of the antithrombin deltaGlu mutant at different stages, and processing of N-linked oligosaccharides highly correlated with the efficiency of ERAD.

A model and theoretical equations are presented to investigate the enantiomeric recognition mechanism of primary amino compounds using an achiral crown ether with cyclodextrin by capillary electrophoresis (CE) and nuclear magnetic resonance (NMR). Association constants were calculated from CE and 1H NMR experiment results on the basis of the model and the equations. The key step of chiral recognition was identified from those values. Using CE analyses of three primary amino compounds [1-(1-naphthyl)ethylamine; 1-aminoindan; 1,2,3,4-tetrahydro-1-naphthylamine], the key step was identified with the equilibrium where the complex of a primary amino compound and 18-crown-6 becomes associated with 2,6-di-O-methyl-beta-cyclodextrin for all the three compounds. From the 1H NMR analyses of 1-(1-naphthyl)ethylamine, the key step was identified with the equilibrium where the complex of 1-(1-naphthyl)ethylamine and 18-crown-6 becomes associated with beta-cyclodextrin.

A novel enantiomeric separation method by capillary electrochromatography with chiral crown ether-bonded negatively charged polyacrylamide gels is presented. Two kinds of chiral crown ether derivatives, (+)-tetraallyl 18-crown-6 carboxylate and (+)-18-crown-6 tetracarboxylic acid 2-allyl ester were synthesized and allowed to covalently bind to a negatively charged polyacrylamide gel, a so-called monolithic stationary phase, respectively. The gel was placed in fused-silica tubing, the walls of which had been activated with a bifunctional reagent to make the resulting gel bind covalently to the inner surface. Enantiomeric separations of 12 primary amino compounds were achieved using these columns and mobile phases of 200 mM triethanolamine-300 mM boric acid buffers with high efficiencies of up to 135000 plates m(-1). Both the within- and between-run reproducibilities of retention time and separation factor were good. The reproducibilities of retention time and separation factor for three different columns prepared from a different batch of monomers were acceptable. The gel-filled capillaries were stable for at least 13 months with intermittent use for 3 months followed by storage at room temperature for 10 months. The result of the optical purity test of alanine-2-naphthylamide is also described.

To identify proteins that interact with HSP47, an endoplasmic reticulum (ER)-resident molecular chaperone, a yeast two-hybrid screening was performed using mouse full-length HSP47 including an N-terminal signal sequence as a bait. Analysis of several positive clones led to the identification and cloning of a novel gene, ubin, encoding a ubiquitin-like protein. Unlike other ubiquitin-like proteins, UBIN was shown to interact with signal sequences of various secretory and ER-luminal proteins, including HSP47, but not interact with signal sequences of mitochondrial targeting in two-hybrid system. The possible function of UBIN will be discussed with regards to novel characteristics of binding to signal sequences for ER targeting., (Copyright 2001 Academic Press.)

Enantiomeric separation by capillary electrochromatography with beta-cyclodextrin-bonded negatively charged polyacrylamide gels was examined. The columns used are capillaries filled with a negatively charged polyacrylamide gel, a so-called monolithic stationary phase, to which allyl carbamoylated beta-CD (AC-beta-CD) derivatives covalently bind. The capillary wall is activated first with a bifunctional reagent to make the resulting gel bind covalently to the inner surface of the fused-silica tubing. Enantiomeric separations of 15 cationic compounds were achieved using the above-mentioned columns and mobile phases of 200 mmol l(-1) Tris-300 mmol I(-1) boric acid buffer (pH 7.0 or 9.0) or 200 mmol l(-1) Tris-300 mmol l(-1) boric acid buffer (pH 7.0) containing an achiral crown ether (18-crown-6). Enantiomeric separations of two neutral compounds were also achieved using 200 mmol l(-1) Tris-300 mmol l(-1) boric acid buffer (pH 9.0) as a mobile phase. High efficiencies of up to 150,000 plates m(-1) were obtained. Both the within- and between-run reproducibilities of retention time and separation factor were good. The reproducibilities of retention time and separation factor for three different columns prepared from a different batch of monomers were acceptable. The gel-filled capillaries were stable for at least 3 months with intermittent use, utilizing the mobile phase of 200 mmol I(-1) Tris-300 mmol I(-1) boric acid buffer (pH 9.0).

A homozygous cross-reacting material negative factor XII-deficient patient with 3% antigen and activity levels of factor XII was screened for the identification of a mutation at the genomic level. Low-ionic strength single-stranded conformation polymorphism (SSCP) analysis and sequence analysis showed that the proband's gene for factor XII had an A-->G substitution at nucleotide position 7832 in exon 3, resulting in a Tyr34 to Cys substitution in the NH2-terminal type II domain of factor XII. We designated this mutation as factor XII Tenri. Mutagenic polymerase chain reaction (PCR), followed by KpnI digestion, showed a homozygous mutation in the proband's gene and heterozygous mutations in his parents and sister. Immunoprecipitation and Western blot analyses of plasma samples from the factor XII Tenri family indicated that the proband had a trace amount of variant factor XII with an apparent molecular mass of 115 kD, which was converted to the normal 80-kD form after reduction, suggesting that factor XII Tenri was secreted as a disulfide-linked heterodimer with a approximately 35-kD protein, which we identified as alpha1-microglobulin by immunoblotting. Pulse-chase experiments using baby hamster kidney (BHK) cells showed that Tenri-type factor XII was extensively degraded intracellularly, but the addition of cystine resulted in increased secretion of the mutant. Using membrane-permeable inhibitors, we observed that the degradation occurred in the pre-Golgi, nonlysosomal compartment and a proteasome appeared to play a major role in this process. On the basis of these in vitro results, we speculate that the majority of the factor XII Tenri is degraded intracellularly through a quality control mechanism in the endoplasmic reticulum (ER), and a small amount of factor XII Tenri that formed a disulfide-linked heterodimer with alpha1-microglobulin is secreted into the blood stream.

The tail bud comprises the caudal extremity of the vertebrate embryo, containing a pool of pluripotent mesenchymal stem cells that gives rise to almost all the tissues of the sacro-caudal region. Treatment of pregnant mice with 100 mg/kg all-trans retinoic acid at 9.5 days post coitum induces severe truncation of the body axis, providing a model system for studying the mechanisms underlying development of caudal agenesis. In the present study, we find that retinoic acid treatment causes extensive apoptosis of tail bud cells 24 h after treatment. Once the apoptotic cells have been removed, the remaining mesenchymal cells differentiate into an extensive network of ectopic tubules, radially arranged around the notochord. These tubules express Pax-3 and Pax-6 in a regionally-restricted pattern that closely resembles expression in the definitive neural tube. Neurofilament-positive neurons subsequently grow out from the ectopic tubules. Thus, the tail bud cells remaining after retinoic acid-induced apoptosis appear to adopt a neural fate. Wnt-3a, a gene that has been shown to be essential for tail bud formation, is specifically down-regulated in the tail bud of retinoic acid-treated embryos, as early as 2 h after retinoic acid treatment and Wnt-3a transcripts become undetectable by 10 h. In contrast, Wnt-5a and RAR-gamma are still detectable in the tail bud at that time. Extensive cell death also occurs in the tail bud of embryos homozygous for the vestigial tail mutation, in which there is a marked reduction in Wnt-3a expression. These embryos go on to develop multiple neural tubes in their truncated caudal region. These results suggest that retinoic acid induces down-regulation of Wnt-3a which may play an important role in the pathogenesis of axial truncation, involving induction of widespread apoptosis, followed by an alteration of tail bud cell fate to form multiple ectopic neural tubes.

Previously, we found the first congenital deficiency of histidine-rich glycoprotein (HRG) in a Japanese woman with thrombosis. To elucidate the genetic basis of this deficiency, we first performed Southern blot analysis and found no gross deletion or insertion in the proband's HRG gene. We then examined the nucleotide sequences of all seven exons of the proband's HRG gene. A single nucleotide substitution, G to A at nucleotide position 429, which mutates Gly85 to Glu in the first cystatin-like domain, was found in exon 3 in 13 of 22 amplified clones. This mutation generates a unique Taq I site. Exon 3 was amplified from the proband, her family members, and 50 unrelated normal Japanese individuals, and Taq I fragmentation was examined. Fragmentation of exon 3 was observed in one allele of the genes from the proband and the family members who also have decreased plasma levels of HRG. Fifty unrelated normal Japanese individuals had a normal HRG gene, indicating that the G to A mutation is not a common polymorphism. To elucidate the identified mutation as a cause for the secretion defect of HRG in the proband's plasma, we constructed and transiently expressed the recombinant Tokushima-type HRG mutant (Gly85 to Glu) in baby hamster kidney (BHK) cells, and examined an intracellular event of the mutant protein. The results showed that only about 20% of the Tokushima-type HRG was secreted into the culture medium, and intracellular degradation of the mutant was observed. Thus, the present study strongly suggests that the HRG deficiency is caused by intracellular degradation of the Gly85 to Glu mutant of HRG in the proband.

Heparin cofactor II (HCII) is a serine proteinase inhibitor in human plasma that rapidly inhibits thrombin in the presence of dermatan sulfate or heparin. To understand the molecular mechanism for HCII deficiency in a patient with reduced circulating HCII antigen, we studied a Japanese patient with type I HCII deficiency who suffered from angina pectoris and coronary artery disease. Polymerase chain reaction (PCR)-based sequence analysis showed that the propositus' gene for HCII (HCII Awaji gene) had a thymine insertion after codon (GAT) for Asp88 in exon II, resulting in a frameshift mutation. Consequently, the abnormal HCII Awaji protein was suggested to have an altered amino acid sequence from position 89 and terminate at 107, thus being composed of the NH2-terminal one fifth of normal HCII and dysfunctional for thrombin inhibition. The molecular weight and pI value of HCII Awaji were calculated to be 12,040 and 3.6, respectively, without posttranslational modification. Mutagenic PCR followed by the Tsp509I digestion showed that a half of the PCR products derived from the propositus and his sister was cleaved, suggesting that his sister also has the same mutant allele. Crossed-immunoelectrophoresis and Western blot analyses of plasma and urine from the the propositus and of plasma from his sister did not provide evidence for the existence of the abnormal HCII, suggesting that little truncated HCII was circulating in the patient's blood. However, stable expression assay using human kidney 293 cells transfected with the expression vector containing cDNA encoding wild-type or Awaji-type HCII showed that mutant as well as wild-type HCII was secreted into culture medium normally. These results suggest that the abnormal HCII Awaji protein is secreted normally, but rapidly degraded in the circulating blood.

Gallic acid, a naturally occurring plant phenol with antioxidative activity, was found to induce cell death in promyelocytic leukemia HL-60RG cells, although many antioxidants are well known to protect the cell from oxidative stress. Morphological and biochemical studies indicated that the gallic acid-induced cell death is apoptosis. Flow cytometric analysis revealed that the apoptosis was not triggered at a specific phase of the cell cycle and that 2 h exposure of gallic acid to HL-60RG cells was enough to induce apoptosis. The inhibitory assay suggested that gallic acid-induced cell death was mediated by reactive oxygen species such as hydrogen peroxide, superoxide anion in addition to Ca2+ ion, calmodulin-dependent enzymes. Structure-activity analysis suggests that gallic acid induces apoptosis in HL-60RG cells, depending on its distinctive feature derived from the structure but not on its antioxidative activity.

To determine whether serological typing of hepatitis C virus correlated with the response to interferon-alpha therapy, hepatitis C virus serotypes were determined by subtype-specific antibody to NS4 polypeptide by enzyme-linked immunosorbent assay in 55 Japanese patients with chronic active hepatitis C who subsequently received recombinant interferon-alpha 2a therapy. Response to interferon-alpha was defined as complete and sustained (n = 12), complete response followed by relapse (n = 26), and no response (n = 17). There was no difference in the clinical biochemical parameters between these patients groups. However, a higher proportion (50.0%) of patients with hepatitis C virus serotype II showed complete and sustained response to interferon-alpha, compared to serotype I (11.1%, p < 0.01). These data indicate that this simple hepatitis C virus serotyping assay is a useful predictor of response to interferon-alpha therapy in patients with chronic hepatitis C virus infection.

Serum of a 67-year-old male patient with smoldering multiple myeloma was shown to contain two monoclonal immunoglobulins, IgG and IgA. For the initial seven months, monoclonal IgG was predominantly elevated. During the next one year and eight months, however, serum concentration of the monoclonal IgA increased, with a concomitant decrease of IgG. N-terminal amino acid sequences of heavy and light chains separated from monoclonal IgG and IgA were analyzed. Both light chains were lambda-type and showed identical amino acid sequences of variable regions. The heavy chains also had the same N-terminal amino acid sequence between IgG and IgA. These results strongly suggest that two monoclonal proteins, IgG and IgA, in this patient were produced by B lymphocytes within a clone and that class switch from IgG to IgA in immunoglobulin production during B cell differentiation has taken place in the clinical course of this case.

A rapid analysis method of strongly acidic amino acids and related compounds by a simple modification of an existing amino acid analyzer is presented. In this method, an anion-exchanger column (2.6 X 150 mm) packed with Hitachi 3013-N resin was developed with 0.2 M citric acid. Complete separation of phosphothreonine, phosphoserine, phosphotyrosine, cysteic acid, homocysteic acid, and glutathionesulfonic acid was achieved within 35 min, with no regeneration of the column being required. Tyrosine-O-sulfate was analyzed by the same column using 2 M sodium acetate buffer, pH 5.5. Performic acid oxidation of a variety of proteins and direct analysis of the products by this system successfully detected cysteine, homocysteine, and/or glutathione bound to proteins through disulfide bonds. This suggest the potential use of the method for analysis of the states of protein thiol groups, especially those of clinically significant mutant proteins where mutation of arginine to cysteine is rather frequently recognized.