Three-Dimensional Proton Beam Writing of Optically Active Coherent Vacancy Spins in Silicon Carbide.

Constructing quantum devices comprises various challenging tasks, especially when concerning their nanoscale geometry. For quantum color centers, the traditional approach is to fabricate the device structure after the nondeterministic placement of the centers. Reversing this approach, we present the controlled generation of quantum centers in silicon carbide (SiC) by focused proton beam in a noncomplex manner without need for pre- or postirradiation treatment. The generation depth and resolution can be predicted by matching the proton energy to the material's stopping power, and the amount of quantum centers at one specific sample volume is tunable from ensembles of millions to discernible single photon emitters. We identify the generated centers as silicon vacancies through their characteristic magnetic resonance signatures and demonstrate that they possess a long spin-echo coherence time of 42 ± 20 µs at room temperature. Our approach hence enables the fabrication of quantum hybrid nanodevices based on SiC platform, where spin centers are integrated into p-i-n diodes, photonic cavities, and mechanical resonators.

Hanging drop monoculture for selection of optimal antioxidants during in vitro maturation of porcine oocytes.

We analysed the effect of three antioxidants that have different functional mechanisms on the in vitro maturation (IVM) of porcine oocytes. Single oocyte monoculture using the hanging drop (HD) system has some advantages such as improving analysis efficiency brought by the smaller number of samples than the number of oocytes cultured in one drop. Direct effects of ligands on single oocytes could also be detected without considering the effects of paracrine factors from other oocytes. After 22 h of pre-culture, denuded oocytes were cultured for 22 h with 0.01 and 0.1 µg/ml of L-carnitine (LC), lactoferrin (LF) or sulforaphane (SF) in the presence/non-presence of oxidant stress induced by H2O2 supplementation to evaluate the reducing effects against oxidative stress on nuclear maturation. As a result, compared with LC and SF, LF showed effective reduction in oxidative stress at a lower concentration (0.01 µg/ml), suggesting that LF is a more effective antioxidant in porcine oocyte IVM. Additionally, LF also increased maturation rate even in culture without H2O2. Our results clearly suggest that the HD monoculture system is useful for screening the substances that affect porcine oocyte culture.

Activation of Lactate Receptor Positively Regulates Skeletal Muscle Mass in Mice.

G protein-coupled receptor 81 (GPR81), a selective receptor for lactate, expresses in skeletal muscle cells, but the physiological role of GPR81 in skeletal muscle has not been fully elucidated. As it has been reported that the lactate administration induces muscle hypertrophy, the stimulation of GPR81 has been suggested to mediate muscle hypertrophy. To clarify the contribution of GPR81 activation in skeletal muscle hypertrophy, in the present study, we investigated the effect of GPR81 agonist administration on skeletal muscle mass in mice. Male C57BL/6J mice were randomly divided into control group and GPR81 agonist-administered group that received oral administration of the specific GPR81 agonist 3-Chloro-5-hydroxybenzoic acid (CHBA). In both fast-twitch plantaris and slow-twitch soleus muscles of mice, the protein expression of GPR81 was observed. Oral administration of CHBA to mice significantly increased absolute muscle weight and muscle weight relative to body weight in the two muscles. Moreover, both absolute and relative muscle protein content in the two muscles were significantly increased by CHBA administration. CHBA administration also significantly upregulated the phosphorylation level of p42/44 extracellular signal-regulated kinase-1/2 (ERK1/2) and p90 ribosomal S6 kinase (p90RSK). These observations suggest that activation of GRP81 stimulates increased the mass of two types of skeletal muscle in mice in vivo. Lactate receptor GPR81 may positively affect skeletal muscle mass through activation of ERK pathway.

Contributions of FASN and SCD gene polymorphisms on fatty acid composition in muscle from Japanese Black cattle.

The fatty acid synthase (FASN) and stearoyl-CoA desaturase (delta-9-desaturase) (SCD) genes affect fatty acid composition. This study evaluated the contributions of polymorphisms of these genes on fatty acid composition in muscle in two different populations: 1189 and 1058 Japanese Black cattle from the Miyagi and the Yamagata populations respectively. We sampled intramuscular fat from the longissimus thoracis muscle in the Miyagi population and from the trapezius muscle in the Yamagata population. The collective contributions of FASN and SCD polymorphisms to total additive genetic variance for oleic acid were 13.46% in the Miyagi population and 16.29% in the Yamagata population and to phenotypic variance were 5.45% and 6.54% respectively. Although the individual effects of FASN and SCD polymorphisms on fatty acid composition were small, overall gene substitution may effectively improve fatty acid composition. In addition, we found that gene polymorphism contributions of fatty acids varied by population even in the same breed.

Soymilk-fermented with Lactobacillus delbrueckii subsp. delbrueckii TUA4408L improves immune-health in pigs.

Lactobacillus delbrueckii subsp. delbrueckii TUA4408L has the ability to grow and ferment soymilk and is able to modulate the innate immune response of intestinal epithelial cells in vitro. These two properties prompt us to evaluate whether the soymilk fermented with the TUA4408L strain can induce beneficial immunomodulatory effects in vivo. For this purpose, pigs were selected as a preclinical model. The studies performed here demonstrated that the L. delbrueckii subsp. delbrueckii TUA4408L-fermented soymilk (TUA4408L FSM) reduced blood markers of inflammation and differentially regulated the expression of inflammatory and regulatory cytokines in the intestinal mucosa. These immunological changes induced by the TUA4408L FSM were associated to an enhanced resistance to pathogenic Escherichia coli and an improved grow performance and meat quality of pigs. The experiments and analysis in our study indicate that the immunobiotic TUA4408L FSM could be an interesting non-dairy functional food to beneficially modulate the intestinal immune system, improve protection against pathogens and reduce inflammatory damage. The preclinical study carried out here in pigs could have a better correlation in humans, compared to a rodent model. However, the clinical relevance of these findings still needs to be confirmed by further research, for example, in controlled human challenge studies.

[How to treat infected pacemaker leads in very elderly patients].

Infection of pacemaker leads is one of the difficult problems after implantation, especially in elderly patients. Three octogenarians were referred to our hospital due to infection of pacemaker leads which had been resistant to antibiotic treatments. The patients were 81 approximately 88 years old including 1 male and 2 females. Primary implantation of pacemaker had been performed 9 months approximately 16 years before and infection had started 7 months approximately 7 years before. The infected leads were partially resected in an 88-year-old woman who had serious dementia and poor nutrition status. The infected leads were completely removed under cardiopulmonary bypass (on-beating) in an 81-year-old woman who had undergone 3 previous surgical treatments. The infected lead was partially removed in an 82-year-old man, but infection was not perfectly controlled. Therefore, the lead was completely removed under cardiopulmonary bypass with cardiac arrest. In conclusion, complete removal of the whole pacemaker system is desirable, while palliative surgery might be an option for very elderly patients with high risks.

Development of an in vitro immunobiotic evaluation system against rotavirus infection in bovine intestinal epitheliocytes.

The bovine intestinal epithelial cell line (BIE cells) expresses the Toll-like receptor (TLR)3 and is able to mount an antiviral immune response after the stimulation with poly(I:C). In the present study, we aimed to further characterise the antiviral defence mechanisms in BIE cells by evaluating the innate immune response triggered by rotavirus (RV) infection. In addition, we attempted to determine whether immunobiotic bifidobacteria are able to confer protection of BIE cells against RV infection by beneficially modulating the antiviral immune response. RV OSU (porcine) and UK (bovine) effectively infected BIE cells, while a significant lower capacity to infect BIE cells was observed for human (Wa) and murine (EW) RV. We observed that viral infection in BIE cells triggered TLR3/RIG-I-mediated immune responses with activation of IRF3 and TRAF3, induction of interferon beta (IFN-ß) and up-regulation of inflammatory cytokines. Our results also demonstrated that preventive treatments with Bifidobacterium infantis MCC12 or Bifidobacterium breve MCC1274 significantly reduced RV titres in infected BIE cells and differentially modulated the innate immune response. Of note, both strains significantly improved the production of the antiviral factor IFN-ß in RV-infected BIE cells. In conclusion, this work provides comprehensive information on the antiviral immune response of BIE cells against RV, that can be further studied for the development of strategies aimed to improve antiviral defences in bovine intestinal epithelial cells. Our results also demonstrate that BIE cells could be used as a newly immunobiotic evaluation system against RV infection for application in the bovine host.

Modulation of porcine intestinal epitheliocytes immunetranscriptome response by Lactobacillus jensenii TL2937.

In order to evaluate probiotic strains applicable for the beneficial immunomodulation of the porcine gut (immunobiotics), we previously developed a porcine intestinal epitheliocyte cell line (PIE cells). Here, transcriptomic studies using PIE cells were performed considering that this information would be valuable for understanding the mechanisms involved in the protective activity of the immunobiotic strain Lactobacillus jensenii TL2937 against intestinal inflammatory damage in pigs. In addition, those studies would provide criteria for selecting biomarkers for the screening of new immunobiotic strains. We performed microarray analysis to investigate the transcriptomic response of PIE cells to the challenge with heat-stable enterotoxigenic Escherichia coli (ETEC) pathogen-associated molecular patterns (PAMPs) and, the changes induced by L. jensenii TL2937 in that response. The approach allowed us to obtain a global overview of the immune genes involved in the response of PIE cells to heat-stable ETEC PAMPs. We observed that L. jensenii TL2937 differently modulated gene expression in ETEC PAMPs-challenged PIE cells. Microarray and RT-PCR analysis indicated that the most remarkable changes in PIE cells transcriptomic profile after heat-stable ETEC PAMPs challenge were observed in chemokines, adhesion molecules, complement and coagulation cascades factors. In addition, an anti-inflammatory effect triggered by TL2937 strain in PIE cells was clearly demonstrated. The decrease in the expression of chemokines (CCL8, CXCL5, CXCL9, CXCL10, and CXCL11), complement (C1R, C1S, C3, and CFB), and coagulation factors (F3) by L. jensenii TL2937 supports our previous reports on the immunoregulatory effect of this strain. These results provided clues for the better understanding of the mechanism underlying host-immunobiotic interaction in the porcine host. The comprehensive transcriptomic profiles of PIE cells provided by our analyses successfully identified a group of genes, which could be used as prospective biomarkers for the screening and evaluation of new anti-inflammatory immunobiotics for the prevention of inflammatory intestinal disorders in pigs.

Enhanced proliferative potential in culture of cells from p53-deficient mice.

Normal somatic cells are endowed with limited doubling potential in culture, and the process of immortalization is an inevitable step in neoplastic transformation of the cells. To examine the roles of p53 in this process, the cells of p53-deficient mice were examined for doubling potential. Fibroblast-like cells from a variety of tissues of these mice proliferated continuously without showing aging or crisis. The aneuploid cells overcome the population with passage, but cloning experiment indicated that chromosomal changes were not essential to this process. The enhanced proliferative potential in culture of cells from the p53-deficient mice was also observed in epithelial cells of lens, mammary glands and seminal vesicles and in neural precursor cells. Proliferation of bone marrow cells in response to stem cell factor was enhanced in long term culture, but not in in vitro colony assay; no permanent cell lines could be obtained. No effects of p53-deficiency were found in proliferation of cardiac muscle cells or hepatocytes.

Cooperation between Otx1 and Otx2 genes in developmental patterning of rostral brain.

Otx1 and Otx2 genes are mouse cognates of a Drosophila head gap gene orthodenticle. The homozygous mutants have previously indicated that Otx2 is essential to development of structures anterior to rhombomere 3, probably reflecting its expression around the early primitive streak stage. Otx2 mutation also exhibits craniofacial defects by haplo-insufficiency. Affected structures correspond to the most anterior and most posterior parts of the Otx2 expression where Otx1 is not, or is only weakly, expressed at the time of brain regionalization. No apparent defects are found in early brain development by the Otx1 mutation, suggesting that the Otx1 and Otx2 functions overlap in the regions where both are expressed. To demonstrate this, the Otx1/Otx2 double heterozygous phenotype was examined in this study. Analyses with molecular markers at 9.5 days post coitus suggested the failure in development of mesencephalon and caudal diencephalon with the expansion of anterior metencephalon. Genes expressed in isthmus exhibited a characteristic lateral stripe normally, although rostrally shifted, except that Fgf8 expression was expanded dorsally. The defects were apparent at the 6-somite stage, but not at the 3-somite stage. Broad Fgf8 expression at the 3-somite stage took place normally, but it did not concentrate into a spot corresponding to future isthmus. The double heterozygous phenotype implicates a previously unsuspected mechanism for development of the mes/metencephalic territory; at the 3- to 6-somite stage Otx1 cooperates with Otx2 to establish the mes/diencephalic domain, allowing for the correct development of isthmus/ rhombomere 1.

Experimental model for MDS-like myelodysplasia in transgenic mice harboring the SV40 large-T antigen under an immunoglobulin enhancer.

The SV40 large T gene under the control of immunoglobulin enhancer induced hyperproliferation of multi-lineage hematopoiesis in transgenic mice. Hence the disease has been considered to be an appropriate experimental model for MDS-like myelodysplasia, sequential pathological changes in the development of the disease are introduced in the report. Huge splenomegaly was the major gross abnormality, which developed with 100% frequency; neither hepato-renal, nor other thymico-lymphatic involvement was common. During the progressive increase in splenic weight, extensive proliferation of multi-lineage hemopoiesis was prominent, although no differences were apparent in the cellular proportions of each hematopoietic element compared with normal spleens, either in flow-cytometric analysis using markers for each subset of hematopoietic elements, or in the histological findings. In the later phases of the disease, the proliferating cell type tended to shift to a variety of single to oligo-lineage hemopoiesis, but the majority of mice still showed the presence of multi-lineage hemopoiesis; histologically, such hemopoiesis was somewhat dysplastic, but had no apparent nature of leukemic infiltration. Several transplantation-assays essentially supported the low neoplastic potential of proliferating cells even in later phase. A long-term observation was made aiming to induce more frequent transition of this abnormal hemopoiesis into a single-lineage neoplasm by transplantation of pre-onset spleen cells, as well as bone-marrow cells from transgenic mice at an early phase of the disease, into lethally irradiated C57BL/6 mice. This trial resulted in a variety of neoplastic growths in the recipients; not only was myelodysplastic hypercellularity seen, but also, single-lineage hemopoietic malignancies, such as B-cell lymphomas/leukemias, histiocytic malignancies, and even myeloid leukemias. The transition from multi-lineage myelodysplasia into single lineage hemopoiesis at some frequency is reminiscent of myelodysplastic syndromes (MDS) in humans. Higher frequency of transition into lymphoid malignancies may be due partly to the immunoglobulin enhancer used as a promoter unit. The results that the SV40 large T antigen was expressed in every proliferating cells, there was no apparent increase in multi-CSFs activity; together with the results of the transplantation assays suggest that the hyperproliferation of the cells is directly induced by the expression of SV40 large T antigen in the hemopoietic cells themselves.

Hemopoietic neoplasms in lethally irradiated mice repopulated with bone marrow cells carrying the human c-myc oncogene: a repopulation assay.

Bone marrow (BM) cells from two transgenic mice carrying the human c-myc oncogene were separately harvested, and each sample was injected into 25 lethally irradiated mice. We observed the contribution of the myc gene to the occurrence of hemopoietic neoplasms in the BM-repopulated mice, establishing a new experimental system for analyzing oncogene expression in the hemopoietic system in vivo. The hybrid gene that was transferred into the original transgenic mice was a combination of the human c-myc gene with a regulatory unit consisting of a murine immunoglobulin-heavy chain with an SV40 early-T promoter gene (Ig/Tp-myc). Among the transgenic lines, the tested BM cells were chosen from two lines that had been low-prone in leukemia; in these lines hemopoietic neoplasms did not appear for greater than or equal 200 days after birth. Lethally irradiated controls received BM cells from litters of transgenic mice that did not carry c-myc. The lifetime incidence of hemopoietic neoplasms was 94% and 91% in the two groups of mice repopulated with myc+ BM. By contrast, only 15% of control mice with myc- BM developed hemopoietic lesions. The incidence of hemopoietic malignancies combined with nonthymic lymphomas and myeloma cases (88% and 65%) was higher in the repopulated mice than the incidence of pre-B cell lymphomas in the original transgenic lines (56%). Thirty-two of the 40 myc+ mice that were examined showed the presence of the transferred gene in either the normal hemopoietic tissue or in the hemopoietic neoplasm. Furthermore, 18 of 22 hemopoietic neoplasms studied by Northern hybridization expressed mRNA from the transgenic gene; in other four neoplasms, expression was weak or absent.

MDS-like experimental myelodysplasia: multilineage abnormal hematopoiesis in transgenic mice harboring the SV40 large T antigen under an immunoglobulin enhancer.

The SV40 large T gene under the control of immunoglobulin enhancer induced hyperproliferation of multilineage hematopoiesis in transgenic mice. Huge splenomegaly was the major gross abnormality; mice were rather anemic, and neither leukoerythroblastosis nor invasion into tissues such as liver, kidneys or lymph nodes was common. In the latter phases of the disease, the proliferating cell type tended to shift to a variety of single-lineage hematopoiesis, but the majority of mice still showed the presence of multilineage hematopoiesis; such cells were somewhat dysplastic but low in neoplastic potential. A long-term observation by transplantation of the hematopoietic cells into lethally irradiated C57BL/6 mice resulted in a variety of neoplastic growths in the recipients; not only was myelodysplastic hypercellularity seen, but also single-lineage hematopoietic malignancies such as B cell lymphomas/leukemias, histiocytic malignancies and even myeloid leukemias. The disease bore the proliferative feature solely in the spleen and bone marrow, and the transition from multilineage myelodysplasia into single-lineage hematopoiesis at some frequency is reminiscent of myelodysplastic syndromes (MDS) in humans. The results that the SV40 large T antigen was expressed in every proliferating cell, and that there was no apparent increase in any colony-stimulating cytokine(s), together with the results of the transplantation assays, suggested that the hyperproliferation of the hematopoietic cells was a direct consequence of the expression of SV40 large T antigen in these cells themselves.

Influence of oophorectomy on serum levels of sex steroids and bone metabolism and assessment of bone mineral density in lumbar trabecular bone by QCT-C value.

The present cross-sectional study was carried out to determine the influence of oophorectomy (OPX) on serum levels of sex steroids and bone metabolism, as well as bone mineral density (BMD), in OPX subjects in comparison with age- and body size-matched controls. Quantitative computed tomography (QCT) and dual-photon absorptiometry (DPA) demonstrated a remarkable reduction in BMD in OPX subjects. In particular, the QCT of the centrum of vertebral bone (QCT-C) in these subjects was no more than 69.33 +/- 3.40% (X +/- SEM) of the control value, and this parameter was much lower than the QCT integral (QCT-I) value of total lumbar vertebrae. This means that BMD decreases specifically in spongy portions after OPX. The serum level of estrone (E1) was significantly lower in OPX subjects than in controls. The hormonal action of E1 on target organs has been thought to be only one-third of that of estradiol (E2), but the marked reduction in serum E1 level seemed to be a significant cause of the reduction in BMD. The serum level of androstenedione (delta 4) significantly decreased in OPX subjects and appeared to affect bone metabolism negatively. Both bone formation and bone resorption were found to be stimulated following OPX, but the rate of bone resorption was found to be higher than that of bone formation: there was an imbalance between bone formation and bone resorption in OPX subjects. However, it was not possible to prove a relationship between Ca regulating hormone and this phenomenon. In conclusion, the QCT-C value reflects the changes in spongy vertebral BMD more sensitively than the QCT-I value or DPA.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical structure and biological activity of a lipid A component from Helicobacter pylori strain 206.

The chemical structure of a lipid A, which was obtained as a minor component from lipopolysaccharide of Helicobacter pylori strain 206-1, was determined to be a glucosamine beta-(1 -6) disaccharide 1-(2-aminoethyl)phosphate acylated by (R)-3-hydroxyoctadecanoic acid, (R)-3- hydroxyhexadecanoic acid, and (R)-3-(octadecanoyloxy)octadecanoic acid at the 2-, 3- and 2'-positions, respectively. Compared with the other major lipid A from the same strain, which was previously reported [Suda Y, Ogawa T, Kashihara W et al. Chemical structure of lipid A from Helicobacter pylori strain 206-1 lipopolysaccharide. J Biochem 1997; 121: 1129--1133], the structure was very similar with one exception. An (R)-3-hydroxyhexadecanoic acid was present at the 3-position of the novel lipid A component. The structure is apparently identical to one of the proposals by Moran et al. [Moran AP, Lindner B, Walsh EJ. Structural characterization of the lipid A component of Helicobacter pylori rough- and smooth-form lipopolysaccharides. J Bacteriol 1997; 179: 6453--6463], who concluded the same structure as the so-called major lipid A from the H. pylori strain NCTC 11637 but without isolating a homogeneous component. The endotoxic properties and pro-inflammatory cytokine-inducing activities of this novel tetra-acyl type lipid A were lower than those of previously reported major tri-acyl type lipid A.

Differences in axial bone mineral density, serum levels of sex steroids, and bone metabolism between postmenopausal and age- and body size-matched premenopausal subjects.

The present study was designed to investigate the differences in serum levels of sex steroids, bone metabolism, and bone mineral density (BMD) between 22 premenopausal subjects and 21 age- and body size-matched postmenopausal subjects. Dual photon absorptiometry (DPA) demonstrated total body BMD significantly decreased in postmenopausal subjects. It was also found that not only spinal BMD as assessed by DPA, but also total third lumbar BMD and third lumbar trabecular BMD as measured by quantitative computed tomography (QCT) significantly decreased in these subjects. In postmenopausal subjects, the urinary-hydroxyproline/creatinine ratio increased significantly, which suggested that bone resorption had been stimulated, and serum levels of alkaline phosphatase (ALPase) and osteocalcin also significantly increased, which suggested a stimulation of bone formation. Consequently, the reduction in BMD was thought to result from a negative balance of bone resorption versus bone formation, or a high turnover of bone metabolism. Androgens were not confirmed to participate in these changes in bone metabolism, but the reduction in serum levels of estradiol, as a major factor, and the reduction in serum levels of estrone, as a participating factor, were assumed to result in an increase in bone resorption in excess of bone formation.

Decreased bone mineral density associated with early menopause progresses for at least ten years: cross-sectional comparisons between early and normal menopausal women.

To establish whether early onset of menopause carries an increased risk of osteoporosis, we compared the bone mineral density (BMD) of the second to fourth lumbar vertebrae (L2-4) between 18 women who had menopause before 43 years of age (early menopause group) and 19 women who had menopause after reaching 43 years of age (normal menopause group). Serum levels of calcium, phosphorus, calcitonin, intact parathyroid hormone, luteinizing hormone (LH), follicle-stimulating hormone (FSH), estradiol (E2), and alkaline phosphatase activity were measured, and urine samples were analyzed to derive calcium/creatinine, hydroxyproline/creatinine, pyridinoline/creatinine, and deoxypyridinoline/creatinine (D-Pyr/Cr) ratios. Mean BMD was significantly lower in the early menopause group than in the normal menopause group, and individual BMD values in about half of the subjects in the former group were below the fracture threshold for Japanese women. Serum concentrations of LH, FSH, and E2 were slightly, but not significantly, lower in the early menopause group than in the normal menopause group. The D-Pyr/Cr ratio was significantly higher in the early menopause group than in the normal menopause group. There was no correlation between L2-4 BMD and age or the number of years after menopause in the normal menopause group, but both age and the number of years after menopause were negatively correlated with L2-4 BMD in the early menopause group. These results indicate that BMD in women who have early menopause continues to decline for up to 10 years, and that menopause and aging increase the risk of osteoporosis.

Structural feature of the major but not cytokine-inducing molecular species of lipoteichoic acid.

Previously, lipoteichoic acid (LTA) of Enterococcus hirae was found to exhibit definite cytokine-inducing activity but synthetic specimens which share the fundamental structural principles proposed for LTA had no corresponding activity. We also showed recently that several minor components totally less than 5% of the LTA fraction from E. hirae ATCC 9790 possessed the activity, whereas the major component (over 90%) did not [Suda, Y., Tochio, H., Kawano, K., Takada, H., Yoshida, T., Kotani, S., and Kusumoto, S. (1995) FEMS Immun. Med. Microbiol. 12, 97-112]. In the present study, the structure of the major component of LTA was studied in an attempt to elucidate the reason for the lack of the activity in the synthetic compounds. The major component of the LTA was first digested by hydrofluoric acid hydrolysis to cleave phosphodiester linkages present. The hydrolysis products were separated and characterized by means of NMR and MS. The linkage positions of the original phosphodiesters were determined from the NMR spectra of an alkali-treated product without hydrofluoric acid degradation. The compound was proved to consist of 1,3-linked poly(glycerophosphate) and a lipid anchor, Glc(alpha1-2)Glc(alpha1-3)acyl(2)Gro, the former being linked to the 6-position of the distal glucose of the latter. The 2-position of the glycerol residues in the glycerophosphate part were substituted by oligoglucose esterified partially with alanine. The gross structure elucidated here thus coincides with the previous conclusion described by Fischer [Fischer, W. (1990) in Glycolipids, Phosphoglycolipids and Sulfoglycolipids (Kates, M., ed.) pp. 123 234, Plenum Press, New York]. Thus, the molecular species with this so-called "LTA structure" is not responsible for the cytokine-inducing activity.

Chemical structure of lipid A from Helicobacter pylori strain 206-1 lipopolysaccharide.

The chemical structure of a novel lipid A, which was obtained as a major component from lipopolysaccharide of Helicobacter pylori strain 206-1, was determined to be a glucosamine beta(1-6) disaccharide 1-(2-aminoethyl)phosphate acylated by (R)-3-hydroxyoctadecanoic acid and (R)-3-(octadecanoyloxy)octadecanoic acid at the 2- and 2'-position, respectively. The absence of a phosphoryl group at the 4'-position and fatty acyl groups at the 3- and 3'-position, and the stoichiometric presence of 2-aminoethyl phosphate at the 1-position are unique features, distinguishing it from the lipid A of enterobacteria.

A novel crosslinking reagent and its application for the detection and isolation of heparin-binding protein(s) on the platelet surface.

A new hetero-bifunctional photo crosslinking reagent, 2-(4-azidoanilyl)-4-(4-azabicyclo-[2,2, 2]hexylammonio)-6-morpholino-1,3,5-triazine chloride, was designed to detect and isolate heparin-binding protein(s) that may act as heparin-receptor(s) on the platelet surface. In a preliminary study using ethanol as a model substrate, the reagent was shown to react with the alcoholic hydroxy group under mild conditions and its crosslinking photoreactivity was high. The reagent effectively formed similar covalent bonds with heparin, while preserving its anticoagulant anti-Xa activity. [3H]Heparin labeled with this reagent crosslinked to antithrombin III very specifically but not to ovalbumin, as analyzed by the Bio-imaging Analyzer System (BAS, Fuji Photo Film, Tokyo). Affinity crosslinking of [3H]heparin was then used to detect heparin-binding proteins on the surface of intact platelets. Several discrete protein bands were detected by the BAS-imaging of SDS-PAGE.