N-Glycan structures of squid rhodopsin.

To determine the glycoforms of squid rhodopsin, N-glycans were released by glycoamidase A digestion, reductively aminated with 2-aminopyridine, and then subjected to 2D HPLC analysis [Takahashi, N., Nakagawa, H., Fujikawa, K., Kawamura, Y. & Tomiya, N. (1995) Anal. Biochem.226, 139-146]. The major glycans of squid rhodopsin were shown to possess the alpha1-3 and alpha1-6 difucosylated innermost GlcNAc residue found in glycoproteins produced by insects and helminths. By combined use of 2D HPLC, electrospray ionization-mass spectrometry and permethylation and gas chromatography-electron ionization mass spectrometry analyses, it was revealed that most (85%) of the N-glycans exhibit the novel structure Manalpha1-6(Manalpha1-3)Manbeta1-4GlcNAcbeta1-4(Galbeta1-4Fucalpha1-6)(Fucalpha1-3)GlcNAc.     

3-Aminobenzamide,a potent inhibitor of poly (ADP-ribose) polymerase,causes a rapid death of HL-60 cells cultured in serum-free medium

HL-60 cells transferred from serum-supplemented to serum-free culture medium initially bound to culture plate tightly and then released from the plate on increasing the culture time and resumed exponential growth after about 8 h lag. At the initial stage of the culture, the cells became extremely sensitive to 3-aminobenzamide, a potent inhibitor of poly (ADP-ribose) polymerase, and, at 1 mM, 80 to 90% of the cells were lysed within 20 h, whereas the inhibitor was totally ineffective on the cell growth in serum-supplemented medium at the concentration. Non-inhibitory analogs of the inhibitor were ineffective. Assay of poly(ADP-ribose) polymerase activity in permeable cells indicated that a transient activation of the enzyme occurred during the culture in serum-free medium (the maximum activation was observed at 8 h of the culture). The cells conditioned in serum-free medium for 24 h acquired significant resistancy to the inhibitor. A low concentration of fibronectin (5 to 10/ml) and a relatively high concentration of bovine serum albumin (0.5 to 1 mg/ml) effectively blocked the cell attachment to plate and also the 3-aminobenzamide-induced cell lysis. These results suggest that poly(ADP-ribose) polymerase is involved in a process essential for HL-60 cells to adapt to a serumdeprived growth condition.     

Effect of 12 weeks of yoga training on the somatization, psychological symptoms, and stress-related biomarkers of healthy women

Background

Previous studies have shown that the practice of yoga reduces perceived stress and negative feelings and that it improves psychological symptoms. Our previous study also suggested that long-term yoga training improves stress-related psychological symptoms such as anxiety and anger. However, little is known about the beneficial effects of yoga practice on somatization, the most common stress-related physical symptoms, and stress-related biomarkers. We performed a prospective, single arm study to examine the beneficial effects of 12 weeks of yoga training on somatization, psychological symptoms, and stress-related biomarkers.

Methods

We recruited healthy women who had no experience with yoga. The data of 24 participants who were followed during 12 weeks of yoga training were analyzed. Somatization and psychological symptoms were assessed before and after 12 weeks of yoga training using the Profile of Mood State (POMS) and the Symptom Checklist-90-Revised (SCL-90-R) questionnaires. Urinary 8-hydroxydeoxyguanosine (8-OHdG), biopyrrin, and cortisol levels were measured as stress-related biomarkers. The Wilcoxon signed-rank test was used to compare the stress-related biomarkers and the scores of questionnaires before and after 12 weeks of yoga training.

Results

After 12 weeks of yoga training, all negative subscale scores (tension-anxiety, depression, anger-hostility, fatigue, and confusion) from the POMS and somatization, anxiety, depression, and hostility from the SCL-90-R were significantly decreased compared with those before starting yoga training. Contrary to our expectation, the urinary 8-OHdG concentration after 12 weeks of yoga training showed a significant increase compared with that before starting yoga training. No significant changes were observed in the levels of urinary biopyrrin and cortisol after the 12 weeks of yoga training.

Conclusions

Yoga training has the potential to reduce the somatization score and the scores related to mental health indicators, such as anxiety, depression, anger, and fatigue. The present findings suggest that yoga can improve somatization and mental health status and has implications for the prevention of psychosomatic symptoms in healthy women.

Trial registration

University Hospital Medical Information Network (UMIN CTR) UMIN000007868.     

Some human B and T cell epitopes of bovine serum albumin,the major beef allergen

Bovine serum albumin (BSA) is the major beef allergen. Since IgE and T cell recognitions are central to the specific immune response to allergens, the identification and immunologic characterization of B and T cell epitopes of BSA represent important steps in the development of treatments for beef allergy. Prior to our experiments, we hypothesized that BSA-specific antibodies and T cells react primarily with sequential epitopes in which the amino acid sequences differ greatly between bovine and human albumin. To clarify this hypothesis, 16 peptides corresponding to such regions were synthesized as candidate epitopes. Among them, at least two regions, aa336-345 and aa451-459, were found to be B cell (IgE-binding) epitopes. In inhibition ELISA experiments, EYAV (aa338-341) and LILNR (aa453-457) bound to patient IgE antibodies and were found to be the cores of the IgE-binding epitopes. Three regions, DDSPDLPKLKPDPNTLC (aa107-123), PHACYTSVFDKLKHLVDEP (aa364-382), and LSLILNRLC (aa451-459), were found to induce T cell proliferation in more than half of the patients tested. Of interest was that these three regions were also recognized by B cells. Information concerning human B and T cells epitopes can contribute greatly to the elucidation of the etiology of beef allergy.     

Characterization of cyanobacteriochrome TePixJ from a thermophilic cyanobacterium Thermosynechococcus elongatus strain BP-1

A putative photoreceptor gene, TepixJ, of a thermophilic cyanobacterium is homologous to SypixJ1 that mediates positive phototaxis in the unicellular motile cyanobacterium Synechocystis sp. PCC 6803. The putative chromophore-binding GAF domain of TePixJ protein was overexpressed as a fusion with a polyhistidine tag (His-TePixJ_GAF) in Synechocystis cells and isolated to homogeneity. The photoreversible conversion of His-TePixJ_GAF showed peaks at 531, 341 and 266 nm for the green light-absorbing form (Pg form), and peaks at 433 and 287 nm for the blue light-absorbing form (Pb form). At 77K, the Pg form fluoresced at 580 nm, while the Pb form did not emit any fluorescence. Mass spectrometry of the tryptic chromopeptide demonstrated that a phycocyanobilin isomer binds to the conserved cysteine at ring A via a thioether bond. It is established that TePixJ and SyPixJ1 are novel photoreceptors in cyanobacteria ('cyanobacteriochromes') that are similar, but distinct from the phytochromes and bacteriophytochromes.     

Quantitative determination of sulfated glycopeptide by two-dimensional electrophoresis on cellulose acetate membrane

Quantitative determination of the sulfated glycoproteins present in tissue and secretion fluid was performed. After digestion of the specimen with pronase in order to convert glycoproteins to glycopeptides, the sulfated glycopeptides were separated from a mixture of acidic glycans (glycosaminoglycans, sialoglycopeptides and sulfated glycopeptides) by two-dimensional electrophoresis on cellulose acetate membrane [(1986) J. Biochem. Biophys. Methods 12, 239-246]. After staining with alcian blue, the spot of sulfated glycopeptide on the cellulose acetate membrane was cut out, and then only the dye bound to the sulfated glycopeptide was extracted with a 5% cetylpyridinium chloride solution at 100 degrees C for 15 min. The extract was then measured by absorbance at 615 nm using an authentic sulfated glycopeptide as a standard. This method facilitated the determination of sulfated glycopeptides, which were separated from other acidic glycans, within the range 0-25 micrograms.     

Cyanobacterial phytochrome-like PixJ1 holoprotein shows novel reversible photoconversion between blue- and green-absorbing forms

The gene, pixJ1 (formerly pisJ1), is predicted to encode a phytochrome-like photoreceptor that is essential for positive phototaxis in the unicellular cyanobacterium Synechocystis sp. PCC 6803 [Yoshihara et al. (2000) Plant Cell Physiol. 41: 1299]. The PixJ1 protein was overexpressed as a fusion with a poly-histidine tag (His-PixJ1) and isolated from Synechocystis cells. A zinc-fluorescence assay suggested that a linear tetrapyrrole was covalently attached to the His-PixJ1 protein as a chromophore. His-PixJ1 showed novel photoreversible conversion between a blue light-absorbing form (Pb, lambdaAmax=425-435 nm) and a green light-absorbing form (Pg, lambdaAmax=535 nm). Dark incubation led Pg to revert to Pb, indicative of stability of the Pb form in darkness. Red or far-red light irradiation, which is effective for photochemical conversion of the known phytochromes, produced no change in the spectra of Pb and Pg forms. Site-directed mutagenesis revealed that a Cys-His motif in the second GAF domain of PixJ1 is responsible for binding of the chromophore. Possible chromophore species are discussed with regard to the novel photoconversion spectrum.     

The Constituents of the Essential Oil from Japanese Quince Fruit,Cydonia oblonga Miller

The biosyntheses of sescandelin (1) and sescandelin B (2) were studied by feeding of [¹³C]labelled precursors to Sesquicilium candelabrum. The labelling pattern of those compounds enriched from the [1-¹³C], [2-¹³C], and [l,2-¹³C]acetates, and from the [¹³C]formate showed that both compounds were derived from a pentaketide chain and a C₁ unit. The isocoumarin skeleton of 1 and 2 is considered to have been formed by the cyclization of a pentaketide chain and a C₁ unit, and of a pentaketide, respectively.     

DBC2 is essential for transporting vesicular stomatitis virus glycoprotein

DBC2 is a tumor suppressor gene linked to breast and lung cancers. Although DBC2 belongs to the RHO GTPase family, it has a unique structure that contains a Broad-Complex/Tramtrack/Bric a Brac (BTB) domain at the C terminus instead of a typical CAAX motif. A limited number of functional studies on DBC2 have indicated its participation in diverse cellular activities, such as ubiquitination, cell-cycle control, cytoskeleton organization and protein transport. In this study, the role of DBC2 in protein transport was analyzed using vesicular stomatitis virus glycoprotein (VSVG) fused with green fluorescent protein. We discovered that DBC2 knockdown hinders the VSVG transport system in 293 cells. Previous studies have demonstrated that VSVG is transported via the microtubule motor complex. We demonstrate that DBC2 mobility depends also on an intact microtubule network. We conclude that DBC2 plays an essential role in microtubule-mediated VSVG transport from the endoplasmic reticulum to the Golgi apparatus.