Native polysomes of Saccharomyces cerevisiae in liquid solution observed by atomic force microscopy

The native polysomes of Saccharomyces cerevisiae were visualized in liquid solution by atomic force microscopy without external contrasting, such as shadowing and negative staining. This study showed native polysomes as lined particle with a height of ca. 27 nm, which is agreement with the height of 80S ribosomes in previous study. We found a small subparticle, located in a ring-shape or at the end of a linear structure, and visualized mRNA chains between adjacent ribosomes. Although the structures of polysomes have been studied for decades, it has remained difficult to visualize the native three-dimensional form. By the observation in liquid solution, we temporarily stopped the translation using an antibiotic to presenting the native three-dimensional structure and function of the polysomes. Our results provide not only new findings on native eukaryotic polysomes, but also great potential to visualize the influence of various environmental conditions on polysomes.     

Prevalence of antibodies against Simkania negevensis in a healthy Japanese population determined by the microimmunofluorescence test

Simkania negevensis has been associated with bronchiolitis in infants and community-acquired pneumonia in adults. Reports of exposure to this microorganism are only available from Israel, North America and Western Europe. Currently, no standard method for diagnosis of S. negevensis infection has been established nor have prevalence rates been shown in Japan. For the first time we demonstrated the ability of the microimmunofluorescence (MIF) test to detect S. negevensis-specific immunoglobulin G and exposure to S. negevensis in Japan. The positive rate in healthy volunteers was 4.3% (25/588), with rates increasing with age. Results indicate the usefulness of the MIF test as a serological method for detecting S. negevensis-specific antibodies. A standard serological test for infection with S. negevensis is needed.     

Inactivation of NF1 in CNS causes increased glial progenitor proliferation and optic glioma formation

The gene responsible for neurofibromatosis type 1 (NF1) encodes a tumor suppressor that functions as a negative regulator of the Ras proto-oncogene. Individuals with germline mutations in NF1 are predisposed to the development of benign and malignant tumors of the peripheral and central nervous system (CNS). Children with this disease suffer a high incidence of optic gliomas, a benign but potentially debilitating tumor of the optic nerve; and an increased incidence of malignant astrocytoma, reactive astrogliosis and intellectual deficits. In the present study, we have sought insight into the molecular and cellular basis of NF1-associated CNS pathologies. We show that mice genetically engineered to lack NF1 in CNS exhibit a variety of defects in glial cells. Primary among these is a developmental defect resulting in global reactive astrogliosis in the adult brain and increased proliferation of glial progenitor cells leading to enlarged optic nerves. As a consequence, all of the mutant optic nerves develop hyperplastic lesions, some of which progress to optic pathway gliomas. These data point to hyperproliferative glial progenitors as the source of the optic tumors and provide a genetic model for NF1-associated astrogliosis and optic glioma.     

Matrix metalloproteinase-9 in macrophages induces thymic neovascularization following thymocyte apoptosis

Matrix metalloproteinase-9 (MMP-9) has been implicated in the degradation of the extracellular matrix in a variety of physiological and pathological processes. We found that MMP-9 expression in thymuses of BALB/c mice that had been injected with anti-CD3 Ab to induce thymocyte apoptosis was increased both at mRNA and protein levels. Macrophages are shown to be the principal stromal cells responsible for phagocytosis of dying thymocytes, and macrophages were found to constitutively express MMP-9. The activity of plasmin, which is known as one of the activators for MMP-9, was increased in the thymuses with MMP-9 activation. Binding of Ab HUIV26, which recognizes a cryptic epitope on collagen type IV following proteolytic cleavage, was found to be reduced in MMP-9 knockout mice, suggesting that collagen type IV is a substrate of MMP-9. Although the formation of thymic neovessels was found following thymocyte apoptosis, it was diminished in anti-CD3 Ab-injected MMP-9 knockout mice. In vivo administration of Ab HUIV26 resulted in a reduction of thymic neovascularization. After clearance of apoptotic thymocytes, the number of macrophages in the thymuses was decreased, and this decrease was delayed by blocking of HUIV26 epitope. Taken together, our results suggest that MMP-9 expression in macrophages mediates degradation of collagen type IV and facilitates their migration from the thymus after clearance of apoptotic thymocytes. These studies demonstrate a potential role of macrophage MMP-9 in the remodeling of thymic extracellular matrix following thymocyte apoptosis.     

Recognition by TNF-alpha of the GPI-anchor glycan induces apoptosis of U937 cells

Tumor necrosis factor-alpha (TNF-alpha) binds to TNF-alpha receptors (TNFR) to produce a hexameric (TNF-alpha)(3)-(TNFR)(3) structure that stimulates apoptosis. We found by using ELISA that TNF-alpha binds to the glycosylphosphatidylinositol (GPI) anchor glycans of carcinoembryonic antigen, human placental alkaline phosphatase (hAP), and Tamm-Horsfall glycoprotein. These binding abilities were inhibited by 10(-6)M mannose-6-phosphate. Treatment of hAP with mild acid and phosphatase, which releases the N-acetylglucosamine (GlcNAc) beta1 -->phosphate-->6 residue from the GPI-anchor glycan of hAP, abrogated the binding of TNF-alpha to hAP. Thus, TNF-alpha binds to the GlcNAcbeta1-->phosphate-->6Man residue in GPI-anchor glycans. To investigate whether the carbohydrate-binding ability of TNF-alpha is related to its physiological functions, human lymphoma U937 cells were used. TNF-alpha stimulates U937 cell apoptosis in a dose-dependent manner and the presence of mannose-6-phosphate inhibited this. TNF-alpha-dependent tyrosine phosphorylation of several proteins in U937 cells was also diminished by mannose-6-phosphate. Phosphatidylinositol-specific phospholipase C-pretreatment also inhibited this tyrosine phosphorylation. These data suggest that TNF-alpha stimulates U937 cell apoptosis by forming a high-affinity nanomeric (TNF-alpha)(3)-(TNFR)(3)-(GPI-anchored glycan)(3) complex. The GPI-anchored glycoprotein involved remains to be identified.     

A retaining endo-beta-mannosidase from a dicot plant, cabbage

An endo-beta-mannosidase [EC 3.2.1.152, glycoside hydrolase family 2], which hydrolyzes the Manbeta1-4GlcNAc linkage of N-glycans in an endo-manner, has been found in plant tissues [Ishimizu, T., Sasaki, A., Okutani, S., Maeda, M., Yamagishi, M., and Hase, S. (2004) J. Biol. Chem. 279, 38555-38562]. So far, this glycosidase has been purified only from a monocot plant, a lily. Here, an endo-beta-mannosidase was purified from a dicot plant, cabbage (Brassica oleracea), and characterized. The cabbage endo-beta-mannosidase consists of four polypeptides. These four polypeptides are encoded by a single gene, whose nucleotide sequence is homologous to those of the lily and Arabidopsis endo-beta-mannosidase genes. 1H NMR analysis of the stereochemistry of the hydrolysis of pyridylaminated (PA) Manalpha1-6Manbeta1-4GlcNAcbeta1-4GlcNAc showed that the cabbage endo-beta-mannosidase is a retaining glycoside hydrolase, as are other glycoside hydrolase family 2 enzymes. The enzymatic characteristics, including substrate specificity, of the cabbage enzyme are very similar to those of the lily enzyme. These endo-beta-mannosidases specifically act on Man(n)Manalpha1-6Manbeta1-4GlcNAcbeta1-4GlcNAc-PA (n = 0 to 2). These results suggest that the endo-beta-mannosidase is present in at least the angiosperms, and has common roles, such as the degradation of N-glycans.     

Micellization of conjugated chenodeoxy- and ursodeoxycholates and solubilization of cholesterol into their micelles: comparison with other four conjugated bile salts species

Micelle formations of sodium glyco- and taurochenodeoxycholate (NaGCDC and NaTCDC) and sodium glyco- and tauroursodeoxycholates (NaGUDC and NaTUDC) was studied at 308.2 K for their critical micelle concentrations at various NaCl concentrations by pyrene fluorescence probe, and the degree of counterion binding to micelle was determined using the Corrin-Harkins plots. The degree of counterion binding was found to be 0.37-0.38 for chenodeoxycholate conjugates, while the determination of the degree was quite difficult for ursodeoxycholate conjugates. The change of I1/I3 values on the fluorescence spectrum with the conjugate bile salt concentration suggested two steps for their bile salt aggregation. The first step is a commencement of smaller aggregates, the first cmc, and the second one is a starting of stable aggregates, the second cmc. The aggregation number was determined at 308.2 K and 0.15 M NaCl concentration by static light scattering: 16.3 and 11.9 for sodium NaGCDC and NaTCDC, and 7.9 and 7.1 for NaGUDC and NaTUDC, respectively. The solubilization of cholesterol into the bile salt micelles in the presence of coexisting cholesterol phase and the maximum additive concentration (MAC) of cholesterol was determined against the bile salt concentration. The standard Gibbs energy change for the solubilization was evaluated, where the micelles were regarded as a chemical species. The solubilization was stabilized in the order of NaGUDC approximately = NaTUDC < NaTC < NaGC < NaTCDC < NaGCDC < NaTDC < NaGDC, where the preceding results were taken into the order.