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The Mycobacterium tuberculosis Ser/Thr kinase PknB is implicated in the regulation of bacterial cell growth and cell division. The intracellular kinase function of PknB is thought to be triggered by peptidoglycan (PGN) fragments that are recognized by the extracytoplasmic domain of PknB. The PGN in the cell wall of M. tuberculosis has several unusual modifications, including the presence of N-glycolyl groups (in addition to N-acetyl groups) in the muramic acid residues and amidation of d-Glu in the peptide chains. Using synthetic PGN fragments incorporating these diverse PGN structures, we analyzed their binding characters through biolayer interferometry (BLI), NMR spectroscopy, and native mass spectrometry (nMS) techniques. The results of BLI showed that muropeptides containing 1,6-anhydro-MurNAc and longer glycan chains exhibited higher binding potency and that the fourth amino acid of the peptide stem, d-Ala, was crucial for protein recognition. Saturation transfer difference (STD) NMR spectroscopy indicated the major involvement of the stem peptide region in the PASTA-PGN fragment binding. nMS suggested that the binding stoichiometry was 1:1. The data provide the first molecular basis for the specific interaction of PGN with PknB and firmly establish PGNs as the effective ligands of PknB., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cardiolipin (CL) is a phospholipid located in both the eukaryotic mitochondrial inner membrane and the bacterial cell membrane. Some bacterial CLs are known to contain cyclopropane moieties in their acyl chains. Although the CLs are thought to be involved in the innate immune response, there have been few attempts at chemical synthesis of the CLs, and detailed studies of their biological activities are scarce. Thus, we have developed a synthetic route to CLs containing chiral cyclopropane moieties.

Although field-emission scanning electron microscopy (FE-SEM) has proven very useful in biomedical research, the high vacuum required (10 -3 to 10 -7  Pa) precludes direct observations of living cells and tissues at high resolution and often produces unwanted structural changes. We have previously described a method that allows the investigator to keep a variety of insect larvae alive in the high vacuum environment of the electron microscope by encasing the organisms in a thin, vacuum-proof suit, the 'NanoSuit®'. However, it was impossible to protect wet tissues freshly excised from intact organisms or cultured cells. Here we describe an improved 'NanoSuit' technique to overcome this limitation. We protected the specimens with a surface shield enhancer (SSE) solution that consists of glycerine and electrolytes and found that the fine structure of the SSE-treated specimens is superior to that of conventionally prepared specimens. The SSE-based NanoSuit affords a much stronger barrier to gas and/or liquid loss than the previous NanoSuit did and, since it allows more detailed images, it could significantly help to elucidate the 'real' organization of cells and their functions.

A protocol for the direct analysis of the phospholipid composition in the whole body of adult soil nematode, Caenorhabditis elegans (C. elegans), was developed, which combined freeze-cracking of the exoskeletal cuticle and matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS). Biomolecules in the m/z range from 700 to 900 were more effectively detected in the freeze-cracked than from simple frozen adult nematode bodies. Different distribution of biomolecules was observed in a nematode body when the matrix was applied with a sublimation deposition method. The whole-body IMS technique was applied on genetically deficient mutant C. elegans to combine whole-body lipidomics and genetics, by comparing the fatty acid compositions, especially of the phosphatidylcholine (PC) species, between the wild-type and fat-1 mutants, which lack the gene encoding an n-3 fatty acid desaturase. A significant reduction of PC(20:5/20:5) and PC(20:4/20:5) and a marked increase of PC(20:4/20:4), PC(20:3/20:4), and PC(20:3/20:3) were detected in the fat-1 mutants in positive ion mode. In addition, phospholipid compositions other than PCs were analyzed in negative ion mode. A loss of a possible phosphatidylinositol (PI) with 18:0/20:5 and a compensative accumulation of putative PI(18:0/20:4) were detected in the fat-1 mutants. In conclusion, the whole-body MALDI-IMS technique is useful for the profiling of multiple biomolecules in C. elegans in both intra- and inter-individual levels.

Cryoablation is therapeutically applied for various disorders in several organs, and skin diseases are typical targets as this cryotherapy has been widely used for viral warts, benign tumors, and actinic keratosis. The main mechanisms of cryoablation consist of direct freezing effect on skin constituents, thrombosis formation in microcirculation, and subsequent immunological responses. Among them, however, the immunological mechanism remains unelucidated, and it is an issue how the direct freezing injury induces immunological consequences. We established a mouse cryoablation model with liquid nitrogen applied to the shaved back skin, and used this system to study the immunological excitement. After application of liquid nitrogen, the thermal decrease ratio was -25°C/sec or less and the lowest temperature was less than -100°C, which was sufficient to induce ulceration. Destruction of cornified layer and necrosis of epidermal cells were observed in transmission electron microscopy image, and increased transepidermal water loss and skin permeability were detected by the functional measurements. By flow cytometry, antigen-presenting dendritic cells (DCs), including PDCA1+B220+CD19- plasmacytoid DCs (pDCs) and CD11c+ myeloid DCs, as well as neutrophils and macrophages were increased in subcutaneous tissue. In parallel, the mRNA expressions of interferon α1 which are known as pDC-producing cytokines, was elevated. We also found marked degranulation of mast cells, providing a possibility that released histamine attracts pDCs. Finally, FITC migration assay revealed that pDCs and CD11c+ DCs emigrated from the cryoablated skin to the draining lymph nodes. Our study suggests that cryoablation induces destruction of the barrier/epidermis, accumulation of pDCs and CD11c+ DCs to the skin, and migration of DCs to regional lymph nodes. Viral elements or tumor cell lysates released from damaged keratinocytes may stimulate the DCs, thereby leading to antiviral or antitumor effect.

Although extremely useful for a wide range of investigations, the field emission scanning electron microscope (FE-SEM) has not allowed researchers to observe living organisms. However, we have recently reported that a simple surface modification consisting of a thin extra layer, termed 'NanoSuit', can keep organisms alive in the high vacuum (10(-5) to 10(-7) Pa) of the SEM. This paper further explores the protective properties of the NanoSuit surface-shield. We found that a NanoSuit formed with the optimum concentration of Tween 20 faithfully preserves the integrity of an organism's surface without interfering with SEM imaging. We also found that electrostatic charging was absent as long as the organisms were alive, even if they had not been coated with electrically conducting materials. This result suggests that living organisms possess their own electrical conductors and/or rely on certain properties of the surface to inhibit charging. The NanoSuit seems to prolong the charge-free condition and increase survival time under vacuum. These findings should encourage the development of more sophisticated observation methods for studying living organisms in an FE-SEM., (© 2015 The Author(s) Published by the Royal Society. All rights reserved.)

Scanning electron microscopy (SEM) has made remarkable progress and has become an essential tool for observing biological materials at microscopic level. However, various complex procedures have precluded observation of living organisms to date. Here, a new method is presented by which living organisms can be observed by field emission (FE)-SEM. Using this method, active movements of living animals were observed in vacuo (10(-5)-10(-7) Pa) by protecting them with a coating of thin polymer membrane, a NanoSuit, and it was found that the surface fine structure of living organisms is very different from that of traditionally fixed samples. After observation of mosquito larvae in the high vacuum of the FE-SEM, it was possible to rear them subsequently in normal culture conditions. This method will be useful for numerous applications, particularly for electron microscopic observations in the life sciences., (© The Author 2014. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Multidisciplinary cancer boards (CBs) for making cancer treatment decisions have become popular in many countries; however, the status of radiotherapy in CBs and the influence of CBs on radiotherapy decisions have not been studied. To clarify these issues, we reviewed the minutes of our CBs from February 2010 to March 2012, and we classified planned treatments discussed at the CBs into five categories and analyzed decisions concerning radiotherapy in each category. The fraction of cases for which radiotherapy was recommended was 536/757 (71%). These cases included 478 cases (63%) for which radiation therapy was planned and four cases (0.5%) for which radiation therapy was unexpectedly recommended. On the other hand, radiation therapy was canceled in 21 cases (4%) for which radiation therapy had been planned. This study showed that radiotherapy was discussed in many cases at CBs and that CBs have a great influence on decisions concerning radiotherapy.

Self-standing biocompatible films have yet to be prepared by physical or chemical vapor deposition assisted by plasma polymerization because gaseous monomers have thus far been used to create only polymer membranes. Using a nongaseous monomer, we previously found a simple fabrication method for a free-standing thin film prepared from solution by plasma polymerization, and a nano-suit made by polyoxyethylene (20) sorbitan monolaurate can render multicellular organisms highly tolerant to high vacuum. Here we report thin films prepared by plasma polymerization from various monomer solutions. The films had a flat surface at the irradiated site and were similar to films produced by vapor deposition of gaseous monomers. However, they also exhibited unique characteristics, such as a pinhole-free surface, transparency, solvent stability, flexibility, and a unique out-of-plane molecular density gradient from the irradiated to the unirradiated surface of the film. Additionally, covering mosquito larvae with the films protected the shape of the organism and kept them alive under the high vacuum conditions in a field emission-scanning electron microscope. Our method will be useful for numerous applications, particularly in the biological sciences.

Background: To clarify how a multidisciplinary cancer board (CB) influences treatment decisions., Methods: From March 2010 to June 2011, a total of 475 cases were discussed at our CB and the minutes of the board were reviewed for this study., Results: Of the 475 patients, minor changes in treatment methods were made in 42 patients (9 %) and major changes were made in 28 patients (6 %). Further diagnostic procedures, further publication surveys and reconfirmation of patient's wishes were recommended in 80 patients (17 %). In the 392 patients for whom treatment was recommended, the CB's recommendation was realized in 349 patients (89 %) and was not realized in 20 (5 %) patients., Conclusions: It is obvious that a CB has a great influence on cancer treatment decisions, but the effectiveness of the CB in our hospital should be verified in the future by analyzing treatment outcomes.

Filaggrin protein is synthesized in the stratum granulosum of the skin and contributes to the formation of the human skin barrier. Profilaggrin is cleaved by proteolytic enzymes and converted to functional filaggrin, but its processing mechanism remains not fully elucidated. Kallikrein-related peptidase 5 (KLK5) is a major serine protease found in the skin, which is secreted from lamellar granules following its expression in the stratum granulosum and activated in the extracellular space of the stratum corneum. Here, we searched for profilaggrin-processing protease(s) by partial purification of epidermal extracts and found KLK5 as a possible candidate. We used high performance liquid chromatography coupled with electrospray tandem mass spectrometry to show that KLK5 cleaves profilaggrin. Furthermore, based on a proximity ligation assay, immunohistochemistry, and immunoelectron microscopy analysis, we reveal that KLK5 and profilaggrin co-localize in the stratum granulosum in human epidermis. KLK5 knockdown in normal cultured human epidermal keratinocytes resulted in higher levels of profilaggrin, indicating that KLK5 potentially functions in profilaggrin cleavage.

Most multicellular organisms can only survive under atmospheric pressure. The reduced pressure of a high vacuum usually leads to rapid dehydration and death. Here we show that a simple surface modification can render multicellular organisms strongly tolerant to high vacuum. Animals that collapsed under high vacuum continued to move following exposure of their natural extracellular surface layer (or that of an artificial coat-like polysorbitan monolaurate) to an electron beam or plasma ionization (i.e., conditions known to enhance polymer formation). Transmission electron microscopic observations revealed the existence of a thin polymerized extra layer on the surface of the animal. The layer acts as a flexible "nano-suit" barrier to the passage of gases and liquids and thus protects the organism. Furthermore, the biocompatible molecule, the component of the nano-suit, was fabricated into a "biomimetic" free-standing membrane. This concept will allow biology-related fields especially to use these membranes for several applications.

Aims: The authors investigated the correlation between prolactin concentrations during aripiprazole treatment and various factors, including age, sex, plasma concentrations of aripiprazole and its active metabolite, dehydroaripiprazole, and genetic polymorphisms of dopamine D2 receptor (DRD2) and cytochrome P450(CYP)2D6., Methods: The subjects were 70 inpatients with schizophrenia (36 men and 34 women), receiving fixed doses of aripiprazole (24 mg in 45 cases and 12 mg in 25 cases) for periods of between 2 and 30 weeks. Prolactin concentrations were measured by chemiluminescence immunoassay. Plasma concentrations of aripiprazole and dehydroaripiprazole were measured using liquid chromatography with mass spectrometric detection. The genotypes of Taq1A, -141C Ins/Del DRD2 and CYP2D6 were detected by polymerase chain reaction methods., Results: Prolactin concentrations were significantly higher in women than in men (8.9 ± 7.5 vs 3.4 ± 3.0 ng/mL, P < 0.001). No correlations were found between prolactin concentrations and plasma concentrations of aripiprazole, dehydroaripiprazole or the sum of the two compounds. Prolactin concentrations were not affected by any polymorphism., Conclusion: The present study suggests that only sex plays a significant role in prolactin concentrations during aripiprazole treatment., (© 2012 The Authors. Psychiatry and Clinical Neurosciences © 2012 Japanese Society of Psychiatry and Neurology.)

The CYP2D6*10(*10) allele that causes decreased CYP2D6 activity is present in Asians with a high frequency of approximately 50%. We studied the effects of the *10 allele on the steady-state plasma concentrations of aripiprazole and its active metabolite, dehydroaripiprazole. The subjects were 63 Japanese patients with schizophrenia who had only the wild-type or *10 alleles. Twenty-seven patients were homozygous for the wild-type allele, 31 were heterozygous, and five were homozygous for the *10 allele. All patients had been receiving the fixed doses of aripiprazole for at least 2 weeks. The daily doses were 24 mg (n = 40) and 12 mg (n = 23). No other drugs except biperiden and flunitrazepam were coadministered. Plasma concentrations of aripiprazole and dehydroaripiprazole were measured using liquid chromatography with mass spectrometric detection. The mean ± standard deviation values of concentration/dose ratios of aripiprazole in the patients with zero, one, and two *10 alleles were 9.0 ± 2.9, 12.7 ± 4.4, and 19.0 ± 6.8 ng/mL/mg, respectively, and those values for dehydroaripiprazole were 4.9 ± 1.6, 5.9 ± 1.7, and 5.9 ± 1.9 ng/mL/mg, respectively. The respective values for the sum of aripiprazole and dehydroaripiprazole were 13.9 ± 4.3, 18.6 ± 5.9, and 24.6 ± 8.5 ng/mL/mg. The mean concentration/dose ratios of aripiprazole were significantly (P < 0.01 or P < 0.001) different among the three genotype groups. The values for the sum of aripiprazole and dehydroaripiprazole were significantly higher in patients with one (P < 0.01) and two (P < 0.001) *10 alleles compared with those with zero *10 alleles. This study suggests that the *10 allele plays an important role in controlling the steady-state plasma concentrations of aripiprazole and the sum of aripiprazole and dehydroaripiprazole in Asian subjects.

We report a 60-year-old man with diffuse large B-cell lymphoma harboring both t(3 ; 7)(q27 ; p12) and t(8 ; 14)(q24 ; q32). Although he received six courses of conventional combination chemotherapy plus rituximab, early relapse occurred. Four courses of an intensive salvage regimen and high-dose chemotherapy with autologous peripheral blood stem cell transplantation were performed. The patient has remained in complete remission for over 24 months. This case is noteworthy because both genetic abnormalities are implicated in lymphomagenesis.

Background: The greatest impediment to effective malaria control is drug resistance in Plasmodium falciparum, and thus understanding how resistance impacts on the parasite's fitness and pathogenicity may aid in malaria control strategy., Methodology/principal Findings: To generate resistance, P. berghei NK65 was subjected to 5-fluoroorotate (FOA, an inhibitor of thymidylate synthase, TS) pressure in mice. After 15 generations of drug pressure, the 2% DT (the delay time for proliferation of parasites to 2% parasitaemia, relative to untreated wild-type controls) reduced from 8 days to 4, equalling the controls. Drug sensitivity studies confirmed that FOA-resistance was stable. During serial passaging in the absence of drug, resistant parasite maintained low growth rates (parasitaemia, 15.5%±2.9, 7 dpi) relative to the wild-type (45.6%±8.4), translating into resistance cost of fitness of 66.0%. The resistant parasite showed an apoptosis-like death, as confirmed by light and transmission electron microscopy and corroborated by oligonucleosomal DNA fragmentation., Conclusions/significance: The resistant parasite was less fit than the wild-type, which implies that in the absence of drug pressure in the field, the wild-type alleles may expand and allow drugs withdrawn due to resistance to be reintroduced. FOA resistance led to depleted dTTP pools, causing thymineless parasite death via apoptosis. This supports the tenet that unicellular eukaryotes, like metazoans, also undergo apoptosis. This is the first report where resistance to a chemical stimulus and not the stimulus itself is shown to induce apoptosis in a unicellular parasite. This finding is relevant in cancer therapy, since thymineless cell death induced by resistance to TS-inhibitors can further be optimized via inhibition of pyrimidine salvage enzymes, thus providing a synergistic impact. We conclude that since apoptosis is a process that can be pharmacologically modulated, the parasite's apoptotic machinery may be exploited as a novel drug target in malaria and other protozoan diseases of medical importance.

The late endosomal marker Rab7 has been long believed to be absent from the phagosome containing Mycobacterium tuberculosis (M.tb) in macrophage, but the detail kinetics remains elusive. Here, we found that Rab7 is transiently recruited to and subsequently released from M.tb phagosomes. For further understanding of the effect of Rab7 dissociation from the phagosome, we examined the localization of lysosomal markers on the phagosome in the macrophage expressing a dominant-negative Rab7. The localization of lysosomal associated membrane protein-2 (LAMP-2) on the phagosome was Rab7-independent, while that of cathepsin D was Rab7-dependent. These results agree with the localization of each lysosomal marker on M.tb phagosome at 6h postinfection-i.e., LAMP-2, but not cathepsin D localized on the majority of M.tb phagosomes. These results suggest that the dissociation of Rab7 from M.tb phagosome is the important process in inhibition of phagolysosome biogenesis.

Unlabelled: The feasibility and efficacy of adriamycin or epirubicin in combination with cyclophosphamide followed by weekly paclitaxel (AC/EC-weekly PAC) as adjuvant chemotherapy for breast cancer was investigated., Patients and Methods: Node-positive breast cancer was treated with AC/ EC-weekly PAC, namely AC at 60/600 mg/m(2) or EC at 90/600 mg/m(2) x4 at three-week intervals, followed by weekly PAC (80 mg/m(2)) x 12, namely four cycles of single weekly administration for three weeks followed by a one-week rest (3 x 4 PAC) or single weekly administration for 12 consecutive weeks (12 PAC)., Results: One hundred and three of 109 consecutive patients enrolled were analyzed, of whom 96 (93.2%) completed the regimen. Grade 3/4 neutropenia occurred in 52.4% receiving AC/EC, and 10.9% of 55 receiving 12 PAC but only 2.1% of 48 receiving 3 x 4 PAC. Neuropathy disorders occurred in more than half receiving PAC, which did not improve after one-week rest in 3 x 4 PAC., Conclusion: AC/EC-weekly PAC is feasible and without serious complications.

Background: Systemically podocytic infolding into the GBM which causes nonargyrophilic holes in the GBM in association with intra-GBM microstructures has been considered as a new pathological entity. However, its pathomechanisms are largely unknown., Methods: We analyzed intra-GBM microstructures in an SLE patient with glomerulopathy associated with podocytic infolding by immunoelectron microscopy for vimentin (a marker for both podocyte and endothelium) and C5b-9 and by 3D reconstruction of transmission electron microscopy (TEM) images by computer tomography method., Results: Immunofluorescent study showed immunoglobulin deposition in a diffuse, capillary pattern; however, electron-dense deposits like stage 3 membranous nephropathy could be found only in some capillary loops by TEM in spite of the systemic existence of podocytic infolding and the intra-GBM microstructures. Three-dimensional reconstructed images of the TEM images revealed that some of the intra-GBM microstructures made connections with the podocyte. The clustered microstructures underneath the podocyte and their surroundings looked as a whole like the degraded part of podocyte in 3D reconstructed images. Immunoelectron microscopy showed that vimentin was positive in most intra-GBM microstructures. C5b-9 was positive along the entire epithelial side of the GBM and in some microstructures, suggesting that the podocytes may be attacked by C5b-9 and that the microstructures may contain C5b-9 bound cellular membranes., Conclusion: Intra-GBM microstructures may be originated mainly from the podocyte. Podotyte and GBM injuries caused by C5b-9 attack to podocytes might contribute in part to podocytic infolding and intra-GBM microstructures in this case.

Schwannomas are tumors arising from the nerve sheath. Because of their infrequent occurrence, nonspecific symptoms, and lack of distinguishing radiologic features, schwannomas are most often diagnosed histologically after surgical excision. We report herein a case of a perinephric, retroperitoneal schwannoma which, after evaluation by computed tomography, magnetic resonance, and angiography, appeared to be a renal cell carcinoma arising from the left kidney. The diagnosis of benign retroperitoneal schwannoma was made by histologic examination and immunohistochemical staining of the excised mass. A practical algorithm for perinephric, retroperitoneal schwannomas is proposed.

The intensity of Galactic cosmic rays is nearly isotropic because of the influence of magnetic fields in the Milky Way. Here, we present two-dimensional high-precision anisotropy measurement for energies from a few to several hundred teraelectronvolts (TeV), using the large data sample of the Tibet Air Shower Arrays. Besides revealing finer details of the known anisotropies, a new component of Galactic cosmic ray anisotropy in sidereal time is uncovered around the Cygnus region direction. For cosmic-ray energies up to a few hundred TeV, all components of anisotropies fade away, showing a corotation of Galactic cosmic rays with the local Galactic magnetic environment. These results have broad implications for a comprehensive understanding of cosmic rays, supernovae, magnetic fields, and heliospheric and Galactic dynamic environments.

Prolactin, a peptide hormone essential for the development and function of reproductive organs, is involved in development of breast, prostate and colorectal cancers. However, the role of prolactin on the carcinogenesis of ovarian carcinomas is unclear. In this study, we show that mRNA of prolactin receptor is expressed in 5 out of 9 ovarian carcinoma cell lines, 15 out of 17 cases of surgical samples and all of normal ovarian surface epithelium, while prolactin transcript is detected only in 1 ovarian carcinoma cell line and in 1 of the surgical samples. For the prolactin receptor-positive ovarian carcinoma cells, exogenous prolactin did not affect the proliferation but markedly inhibited apoptosis. Therefore, actual cell growth was enhanced by prolactin in a dose-dependent manner. The blocking of prolactin receptor by antibody severely impaired the antiapoptotic and growth-promoting effects of prolactin. Interestingly, the cisplatin-induced cell death of the prolactin receptor-positive cells was significantly inhibited by pretreatment with prolactin. These findings indicate a responsiveness of ovarian carcinomas to prolactin and suggest that the prolactin/prolactin receptor system may be a new therapeutic target of ovarian carcinomas., (Copyright 2005 Wiley-Liss, Inc.)

A contig-oriented database for annotation of the rice genome has been constructed to facilitate map-based rice genomics. The Rice Annotation Database has the following functional features: (i) extensive effort of manual annotations of P1-derived artificial chromosome/bacterial artificial chromosome clones can be merged at chromosome and contig-level; (ii) concise visualization of the annotation information such as the predicted genes, results of various prediction programs (RiceHMM, Genscan, Genscan+, Fgenesh, GeneMark, etc.), homology to expressed sequence tag, full-length cDNA and protein; (iii) user-friendly clone / gene query system; (iv) download functions for nucleotide, amino acid and coding sequences; (v) analysis of various features of the genome (GC-content, average value, etc.); and (vi) genome-wide homology search (BLAST) of contig- and chromosome-level genome sequence to allow comparative analysis with the genome sequence of other organisms. As of October 2004, the database contains a total of 215 Mb sequence with relevant annotation results including 30 000 manually curated genes. The database can provide the latest information on manual annotation as well as a comprehensive structural analysis of various features of the rice genome. The database can be accessed at http://rad.dna.affrc.go.jp/.

We report on the solar diurnal variation of the galactic cosmic-ray intensity observed by the Tibet III air shower array during the period from 1999 to 2003. In the higher-energy event samples (12 and 6.2 TeV), the variations are fairly consistent with the Compton-Getting anisotropy due to the terrestrial orbital motion around the Sun, while the variation in the lower-energy event sample (4.0 TeV) is inconsistent with this anisotropy. This suggests an additional anisotropy superposed at the multi-TeV energies, e.g., the solar modulation effect. This is the highest-precision measurement of the Compton-Getting anisotropy ever made.

A screening assay for inhibitory activity against trypsin in skin mucus from 29 species of fishes reveals a wide distribution of trypsin inhibitors in skin mucus and relatively high antitryptic activity in pufferfish of the family Tetraodontidae. Two trypsin inhibitors termed TPTI 1 and 2 were purified to homogeneity from the skin mucus of Takifugu pardalis by salting out, lectin affinity, anion exchange FPLC and gel filtration HPLC. Both inhibitors are acidic glycoproteins, with an apparent molecular mass of 57 kDa in SDS-PAGE, pI below 4 and 1.9% reducing sugar for TPTI 1 and with an apparent molecular mass of 47 kDa in SDS-PAGE, pI 5.2 and 0.8% reducing sugar for TPTI 2. The inhibitors effectively repress the catalytic activity of trypsin and alpha-chymotrypsin, and therefore can be classified as serine protease inhibitors. The inhibitory constants against trypsin were 4.9x10(-8) M for TPTI 1 and 3.9x10(-8) M for TPTI 2. Both inhibitors react with trypsin at a molar ratio of 1:1, although TPTI 1 reversibly inactivates the proteolytic activity of trypsin non-competitively and TPTI 2, competitively. The trypsin inhibitors in the skin mucus of T. pardalis may function as defense substances to neutralize serine proteases released by invasive pathogens.

The rice species Oryza sativa is considered to be a model plant because of its small genome size, extensive genetic map, relative ease of transformation and synteny with other cereal crops. Here we report the essentially complete sequence of chromosome 1, the longest chromosome in the rice genome. We summarize characteristics of the chromosome structure and the biological insight gained from the sequence. The analysis of 43.3 megabases (Mb) of non-overlapping sequence reveals 6,756 protein coding genes, of which 3,161 show homology to proteins of Arabidopsis thaliana, another model plant. About 30% (2,073) of the genes have been functionally categorized. Rice chromosome 1 is (G + C)-rich, especially in its coding regions, and is characterized by several gene families that are dispersed or arranged in tandem repeats. Comparison with a draft sequence indicates the importance of a high-quality finished sequence.

Sitafloxacin (STFX) hydrate, an antimicrobial agent, is photo-labile in aqueous solutions. The photodegradation rates (k) in neutral solutions were higher than those observed in acidic and alkaline solutions and maximum at the maximum absorption wavelength of STFX. The structures of photodegradation products were elucidated as 7-[7-amino-5-azaspiro[2.4]heptan-5-yl]-6-fluoro-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid and 1-(1-amino-2-[16-fluoro-1-(2-fluoro-1-cyclopropyl)-1,4-dihydro-4-oxo-3-quinolin-7-yl]-amino]ethyl)cyclopropanecarbaldehyde. This implies that dechlorination is the key step in the photodegradation of STFX. The effect of halide ions on the photodegradation of STFX was estimated by observing the increments in the photostability of STFX with the addition of chloride ions. In contrast, in the presence of bromide ions, instead of increased photostability of the STFX rate, a new photodegradation product in the presence of bromide ion was observed. The structure of this new photodegradation product was an 8-bromo form of STFX, which was substituted for chlorine at the 8-position, so the dissociation of C-Cl bond at the 8-position of STFX was the rate-limiting step in the initial process of the photodegradation. STFX generated .C (carbon centered radical) and .OH (hydroxyl radical) in the process of photodegradation in a pH 4.0 buffer. On the contrary, STFX did not generate C in the presence of chloride ion in a pH 4.0 buffer. The .C was generated and then degraded into the above degradation products by photoirradiation in the absence of chloride ion, but the .C immediately reacted with chloride when it was present. As a result, the C-Cl bond was recovered leading to a possible increase in the apparent photostability.

A case of systemic rhabomyolysis after acetonitrile exposure is reported. A 35-year-old previously healthy man suffered from vomiting, convulsion and consciousness loss 15 hours after exposure to acetonitrile. Since acetonitrile is known to be metabolized into cyanide, antidote therapy against cyanide poisoning was given. On admission, pain and all-over muscle swelling were marked. Although the initial therapy was effective, rhabdomyolysis and then acute renal failure developed. Renal function improved very slowly after six weeks of hemodialysis, but atrophy of the muscles remained. The rhabdomyolysis may have been caused by toxicity of the cyanide itself in combination with hypoxia and convulsion.

Background: Hypertriglyceridemia is associated with decreased HDL-cholesterol (HDL-C) and increased small dense LDL. In addition, small dense LDL is known to be susceptible to oxidation., Methods: We measured LDL particle size, using gradient gel electrophoresis, and malondialdehyde-modified LDL (MDA-LDL), using an ELISA, and investigated the association between triglyceride (TG) concentrations, LDL size, and MDA-LDL., Results: TG concentrations correlated negatively with the predominant LDL size (r = -0.650) and HDL-C concentration (r = -0.556). The relationship between TG concentration and LDL size, evaluated by measuring MDA-LDL, distinguished subgroups derived from four subfractions of TG concentrations and four distribution ranges of LDL size. These experiments indicated that there is a threshold for oxidation susceptibility at an LDL size of 25.5 nm and a TG concentration of 1500 mg/L. To investigate the relationship between LDL size, MDA-LDL concentration, and other lipids (TGs, HDL-C, apolipoprotein B, and total cholesterol), we evaluated them in control subjects and patients with diabetes mellitus or hypertriglyceridemia. When the size range for normal LDL was postulated to be 25.5 < or = phi (LDL diameter) < 26.5 nm, the MDA-LDL concentration was significantly higher in the subgroups of patients with LDL in the size range 24.5 < or = phi < 25.5 nm compared with patients with normal LDL. This result also suggests that the threshold is at a LDL size of 25.5 nm., Conclusion: The threshold for oxidation susceptibility coincided with the point of LDL size separation between the LDL subclass patterns A and B as an atherosclerotic risk.

Objective: We aimed to study the effect of the metallic modified Gianturco-Rosch Z-stent in the management of refractory intrahepatic long-segment biliary strictures with hepatolithiasis., Methods: Six symptomatic patients with hepatolithiasis and coexisting intrahepatic long-segment biliary strictures, who failed to respond to the silastic external-internal biliary stenting, were selected. The metallic modified Gianturco-Rosch Z-stent was placed via percutaneous transhepatic cholangiography at the strictured site. Patients were followed regularly to evaluate for recurrence of cholangitis, stones, or strictures., Results: No complications were observed during the procedures. No recurrent strictures or formed calculi were found in these six patients during follow-up periods of 29 to 64 months. However, cholangitis and intrahepatic biliary muddy sludge occurred at 7 and 30 months in two patients after the placement of the metallic Z-stent. Percutaneous transhepatic cholangioscopy was used to clear sludge completely., Conclusions: Our experience suggests that the metallic stent is a well-tolerated and promising alternative in the management of refractory intrahepatic long-segment biliary strictures with hepatolithiasis. Though biliary sludge may develop, it can be detected and cleared early. Repeated surgery can thus be avoided.

The Tibet experiment, operating at Yangbajing (4300 m above sea level), is the lowest energy air shower array, and the new high-density array constructed in 1996 is sensitive to gamma-ray air showers at energies as low as 3 TeV. With this new array, the Crab Nebula was observed in multi-TeV gamma-rays and a signal was detected at the 5.5 sigma level. We also obtained the energy spectrum of gamma-rays in the energy region above 3 TeV which partially overlaps those observed with imaging atmospheric Cerenkov telescopes. The Crab spectrum observed in this energy region can be represented by the power-law fit dJ&parl0;E&parr0;&solm0;dE=&parl0;4.61+/-0.90&parr0;x10-12&parl0;E&solm0;3 TeV&parr0;-2.62+/-0.17 cm-2 s-1 TeV-1. This is the first observation of gamma-ray signals from point sources with a conventional air shower array using scintillation detectors.

Background: Measurement of HDL-cholesterol (HDL-C) by homogeneous assays with automated analyzers is replacing precipitation methods. However, in this reaction-type assay, interactions between the reagents and lipoproteins remain unknown., Methods: Electron microscopy was used to investigate the reactions in a homogeneous HDL-C assay. Negative staining with 10 g/L uranyl acetate was performed for lipoprotein visualization by electron microscopy. Observations of the interactions between lipoproteins and the reagents of a polyanion-polymer/detergent assay were achieved by cooling the reaction mixture in ice water. This treatment also allowed observation of the time course of the reaction., Results: In the first-reagent reaction (polyanion-polymer), every lipoprotein aggregated almost completely. In the second-reagent reaction (enzymes and detergent), only HDL in the lipoprotein aggregates was selectively resolved and reacted enzymatically. Reagent 1 contains two important substances: polyanion and synthetic polymer. Using x-ray microanalysis, we confirmed that aggregation of lipoproteins in the first reaction occurred through interaction with the phosphotungstate of the polyanion., Conclusion: Electron microscopy morphologically revealed the dynamic reaction in a homogeneous HDL-C assay.

Photodynamic therapy (PDT) is a treatment modality that utilizes a photosensitizing drug activated by laser generated light. PDT is effective for oncologic applications. Many cancer patients have undergone a hematoporphyrin derivative (HpD)-mediated PDT. The HpD showed a side effect causing prolonged cutaneous photosensitivity. But ATX-S10, a new photosensitizer, provides rapid plasma and tissue clearance, comparable photoactivation efficiency, and superior light absorption of visible red. In this study, the tumor rejection mechanisms of PDT using ATX-S10 on HeLa tumors in nude mice were investigated with morphological and fluorometric methods. The mice were intracutaneously inoculated with HeLa cells, 5 x 10(5) or 1 x 10(7) cells. When tumors grew to about 10-12 mm in diameter, mice were intraperitoneally administered ATX-S10, 30 mg/kg, and 2 hours later the ATX-S10 in tumors was indirectly measured by a fluorometric machine, PMA-10 (Hamamatsu Photonics K. K.) and the tumors were irradiated by Optical Parametric Oscillator (Hamamatsu Photonics K. K.) tuned to a wave length at 670 nm, 5 mJ/pulse, 100 J/tumor. Before and after the irradiation, the effective mechanisms of PDT with ATX-S10 were studied by histological and ultrastructural approaches. The results showed occlusive thrombi in the microvasculature of the tumors and tumor cell death. These occlusive thrombi were observed within one hour after PDT at both light and electron microscopy levels, and were more remarkable as time passed after PDT. Therefore, the morphological studies of PDT with ATX-S10 suggested that the rejection mechanisms occurred mainly as a result of the destructive changes of the microvasculature in the tumors first, and secondly or simultaneously, tumor cells were destroyed through necrosis, and finally the tumors were rejected.

We have developed a technique of staining the anterior capsule with a solution of indocyanine green that facilitates performance of the circular continuous capsulorrhexis in eyes with a mature cataract. We compared the results of phacoemulsification and intraocular lens implantation in 10 eyes with the capsule stained with results of 10 eyes having the same procedure with standard circular continuous capsulorrhexis. The results of specular microscopy and laser flare-cell photometry showed no statistically significant differences between the 2 groups. Although the safety of intraocular indocyanine green dye has not yet been definitively established, the findings of this pilot study suggest that it is safe and useful in visualizing the anterior capsule of a mature cataract during cataract surgery.

A sensitive method for the assay of Ca2+, Mg(2+)-dependent endonuclease was developed. The assay procedure is composed of two parts: (i) microscale endonuclease digestion of highly polymerized calf thymus DNA and (ii) the quantification of DNA breaks by measuring the activation of poly(ADP-ribose) polymerase, which is known to be activated proportionally to the number of nicks and ends of DNA added in the reaction mixture. This method was approximately 10(5)-fold more sensitive than a conventional DNase assay detecting acid-soluble DNA formation and, thus, the activity of 20 to 100 fg of purified bull seminal Ca2+, Mg(2+)-dependent endonuclease could be reliably measured. Ca2+ and Mg2+ requirements and the response to histone H2B of the endonuclease were also demonstrated by this method. Using this method, the assay of a very small amounts of Ca2+, Mg(2+)-dependent endonuclease in crude extracts of calf thymus chromatin was possible. This method may be applied to other types of endonucleases by modifying the mixture for endonuclease reaction.

Objective: To compare the results, limitations, and complications of the surgical treatment of bilateral hepatolithiasis and intrahepatic biliary strictures with left hepatectomy and without left hepatectomy., Design: Case-controlled study., Setting: Referral center., Patients: During a 12-year period, 103 patients with bilateral hepatolithiasis and intrahepatic biliary strictures underwent surgical treatment. Group A (n = 73) received left hepatic resection (lateral segmentectomy or lobectomy) and postoperative biliary dilatation with residual stone extraction. Group B (n = 30) underwent the same procedures except for left hepatectomy., Interventions: Left lateral segmentectomy or left lobectomy, choledocholithotomy, postoperative cholangioscopic treatments (electrohydraulic lithotripsy, other lithotripsy, lithotomy, balloon dilatation, etc. via T tube or precutaneous transhepatic route)., Main Outcome Measures: Days of hospitalization, incidence of major and minor complications, mortality rates, and the rates of residual stones and stone recurrence were compared., Results: Group A and B had similarly low postoperative 1-month mortality rates of 5.5% and 6.7%, respectively. The main cause of death in both groups was uncontrollable septicemia. The main major complications in group A were intra-abdominal abscess and upper gastrointestinal bleeding; the major complication in group B was massive hemobilia. Group B had a significantly higher overall rate of complications (53.3% vs 23.3%, P < .01) and a longer hospital stay than group A (median, 72 days vs 28 days, P < .03). When complications were classified as major or minor, only minor complications showed a significant difference (30% vs 13.7%, P = .05). After using biliary stricture dilatation and stone extraction, the rate of residual stones in the right lobe was similar in both groups, but patients in group B had a significantly higher rate of residual stones (12.5% vs 0%, P < .02) and stone recurrence in the left lobe (19% vs 0%, P < .003) than those in group A., Conclusions: Partial resection of the left lobe in cases of bilateral hepatolithiasis and biliary strictures can effectively simplify problems in the treatment of bilateral hepatolithiasis and intrahepatic biliary strictures. In addition, not only were surgical complications not increased, but a decrease in complications from postoperative manipulations for stone clearance was noted in our series.

This study examined the survival of orthotopic, vascularized, osteochondral allografts, following 12 weeks of immunosuppression and transfer into a naive, allograft host up to 14 weeks later, and compared the results with those previously reported for similar grafts in a heterotopic position. Knee-joint allografts between DA (donor) and Lewis (recipient) rat strains were assessed by quantitative histology up to 6 months after transplantation, and graft microcirculation was examined by India-ink infusion. Graft repopulation was assessed by re-transferring the graft to a naive, non-suppressed allograft host 12 to 26 weeks after the initial transplantation. Isografts survived for as long as grafts were examined (6 months) and showed good healing of the graft/host bone junction, although long-term isografts showed some deterioration of the growth plate. Non-suppressed allografts rejected within 2 weeks. Allografts in hosts immunosuppressed for 12 weeks remained healthy and healed in a similar manner to the isografts. Following cessation of immunosuppression, allografts progressively deteriorated, with mononuclear cell infiltration apparent in graft bone marrow and muscle in the later stages examined. Transfer to second non-suppressed hosts resulted in rapid rejection of the allografts, indicating that, as shown previously in heterotopic, osteochondral allografts, little or no graft repopulation by host-derived cells had occurred during the protected period in the first host.

Effects of the dopamine-related drug bromocriptine (BCT) on event-related potentials (ERP) were investigated in 18 healthy volunteers. Bromocriptine 2.5 mg or an inactive placebo was administered according to a completely randomized double-blind, cross-over design. The ERP were recorded 3 h after medication was given. Although BCT prolonged the P300 latency, it had no effect on the amplitudes of the ERP components as a whole. Bromocriptine increased the latencies of N100, P200 and P300 in the respective short-latency subject group, and decreased the latency of N200 in the long-latency subject group. It increased the amplitude of N200 in the low-amplitude subject group. It was concluded that the prolongation of P300 latency as a whole and the different responses that take place are dependent on the initial values and were recognized in the effect of a single administration of BCT 2.5 mg. The results of this study are discussed in relation to the law of initial value.

Objective: To investigate the clinical characteristics of a coexisting sharp ductal angulation (< 90 degrees) with biliary stricture and to evaluate the difficulties it imposes in the management of retained or recurrent hepatolithiasis., Design: Case-controlled study., Setting: A referral center., Patients: Eighteen consecutive patients having right-sided hepatolithiasis and a coexisting sharp ductal angulation associated with biliary stricture (group 1) were compared with 84 patients matched with sex, age, and conditions of hepatolithiasis and intrahepatic biliary stricture(s) but no sharp angulated duct (group 2)., Intervention: Postoperative cholangioscopic management (electrohydraulic lithotripsy or other lithotripsy, lithotomy, balloon dilation, biopsy, etc, via T-tube tract or percutaneous transhepatic route)., Main Outcome Measures: Sessions of manipulations, incidence of complications associated with interventions or disease, and mortality were compared., Results: Patients of group 1 needed more sessions of postoperative manipulation of stones and strictures (13.7 +/- 4.2 vs 8.0 +/- 2.3; P < .001). During management, there was a significantly increased vulnerability of severe and/or recurrent cholangitis (66.7% vs 9.5%; P < .001), septic shock (77.8% vs 11.9%; P < .001), liver abscess (55.6% vs 7.1%; P < .001), or massive hemobilia (33.3% vs 7.4%) in group 1 than in group 2. Their risks of coexisting secondary biliary cirrhosis (55.6% vs 9.5%; P < .001) and/or cholangiocarcinoma (16.6% vs 2.4%; P < .04) and mortality (27.8% vs 4.8%; P < .01) were also significantly higher in group 1., Conclusion: Our results suggest that the coexisting sharp ductal angulation with biliary strictures in right-sided hepatolithiasis is a distinct difficult clinical entity in the field of biliary tract calculi.

The effect of 12 weeks of cyclosporin A (CyA) (7 mg/kg) on the survival of vascularized osteochondral allografts between rat strains--Dark Agouti (DA donor) and Lewis (recipient)--was examined up to 6 months after grafting. Grafts were assessed by India-ink infusion to examine their microcirculation, and by quantitative histology. Isografts (Lewis to Lewis) survived at least 25 weeks, but displayed progressive deterioration due to their non-weight bearing position. Rejection controls (allografts with no immunosuppression) showed rejection within 2 weeks. Allografts in immunosuppressed hosts remained healthy for the 12-week period of immunosuppression, but deteriorated progressively during the ensuing 14 weeks, particularly in the muscle, marrow, and growth plates. Graft repopulation by host cells was assessed by transferring grafts into fresh non-suppressed allograft hosts, following 12 to 26 weeks in the first, immunosuppressed host. All grafts were rejected rapidly following the second transfer, indicating that little or no cellular repopulation of the graft had occurred while in the first host.

The effects of treatment in a hydrated autoclave (121 degrees C, 2 atm for 20 min), microwave oven (in water), and simple heating (60 degrees C overnight in distilled water or 90 degrees C for 10 min in ZnSO4) on the stainability of 56 antigens by commercially available antibodies in formalin-fixed paraffin-embedded tissue sections were evaluated. The detectability of nuclear antigens, glycoprotein, lymphocytic surface markers, and chromogranin A was significantly and reproducibly improved by these treatments, whereas the detectability of viral antigens and peptide hormones was attenuated or unchanged. This enhancement includes not only the distinctiveness of the positive staining, but also the number of positive cells, as revealed by comparing serial sections. Among these four heating procedures, microwave heating and autoclaving were more effective than the others on p53, c-erbB-2, and CA125, whereas simple heating was best for smooth-muscle actin (HHF35 and CGA7). Generally the effects of the heating procedures for these antigens were consistent among the cases, but the effects on GFAP varied with the case. The alterations we observed could significantly influence the interpretation of immunohistochemical staining of currently popular tumor markers such as p53 in terms of their prevalence (28% vs 64% in gastric cancer; 36% vs 82% in metastatic liver cancer) and other diagnostically important markers.

Direct nerve-crossing of intercostal nerves from the lateral thorax to the musculocutaneous nerve was performed in 6 patients after spinal nerve root avulsion with brachial plexus palsy. Elbow flexion power was regained well enough to move against gravity and some resistance in all cases. The muscles were examined histochemically 4 (1-9) years after the operation. The intercostally-innervated biceps brachii muscle showed motor predominance of slow-twitch Type 1 fiber regeneration much more than that of fast-twitch Type 2 fiber in 5 of our patients. Our study suggests that the motor nerves of slow-twitch fibers may have priority in peripheral nerve regeneration over those of fast-twitch fibers.

Event-related potentials were recorded in 25 abstinent alcoholics, and 25 gender- and age-matched controls during a two-tone discrimination (odd ball) task. All the subjects were free from medication and dextral. MR images were examined in the alcoholics. The amplitudes of N100, N200 and P300 in the alcoholics were reduced compared with those of the controls. In order to identify morphological changes responsible for ERP abnormalities, linear regression analyses were performed between ERP measures and MRI parameters. The amplitude of N100 was inversely correlated with ventricular size. The amplitudes of P300 were inversely correlated with both ventricular size and width of cortical sulci. It was suggested that the N100 abnormality was related to subcortical structure, and P300 alteration was related to both subcortical and cortical structures in the alcoholics.

The vitreous in both normal eyes of 671 normal subjects was studied biomicroscopically. The results obtained were as follows: 1. In 618 cases of a refractive error less than -3D (group A) and 53 cases of refractive error over -3D (group B), the incidence of posterior vitreous detachment (PVD) in both eyes increased with age and the absence of PVD in both eyes decreased with age. The percentage of cases with PVD in one eye and no PVD in the other eye was less than 22% in all age groups. 2. There was no significant difference in the degree of vitreous liquefaction between the right and the left eyes. The degree of vitreous liquefaction was also age-related. 3. In the cases with PVD in one eye and no PVD in the other eye, a higher incidence of mild vitreous liquefaction was found in group A than in group B. 4. PVD without collapsed vitreous (simple PVD) in both eyes was observed only in group A. 5. Our results indicated that the vitreous findings in both eyes of an individual were almost identical, and PVD may occur with less vitreous liquefaction in group A than in group B.

Prior animal studies of vascularized epiphyseal transfers placed growth plates in 2 bone systems where the independent growth of epiphyses and their response to altered stresses has been difficult to assess. This study assessed growth of vascularized ulnar epiphyses transferred to the ipsilateral humerus of 12-week-old puppies. Growth was permitted by a specially designed extensible plate. Control groups showed that humeral dissection, osteotomy and ostectomy alone do not stimulate growth. In 4 puppies initial growth of the transferred epiphysis was seen but late collapse and formation of bridging callus occurred so that overall humeral length at maturity was not significantly different from control humeri. Physical forces inherent in heterotopic transfer may preclude long term growth of transferred epiphyses particularly in sites of higher relative load. The extensible plate used here may be a useful device in the fixation of transferred epiphyses with growth potential.

A simple and precise method was established for the determination of synephrine in oriental pharmaceutical decoctions containing Evodiae Fructus using high-performance liquid chromatography with sodium dodecyl sulfate (SDS) as an ion-pair reagent. Synephrine was eluted within 25 min without interference from co-existing components using an ODS column and a mixture of water-acetonitrile-SDS-phosphoric acid (70:30:0.5:0.1, v/v/w/v) as a mobile phase.