N-glycans of SREC-I (scavenger receptor expressed by endothelial cells): essential role for ligand binding, trafficking and stability

Scavenger receptor expressed by endothelial cells (SREC-I) mediates the endocytosis of chemically modified lipoproteins such as acetylated low-density lipoprotein (Ac-LDL) and oxidized LDL and is implicated in atherogenesis. We produced recombinant SREC-I in Chinese hamster ovary-K1 cells and identified three potential glycosylation sites, Asn(289), Asn(382) and Asn(393), which were all glycosylated. To determine the function of N-glycans in SREC-I, we characterized SREC-I mutant proteins by intracellular distribution and the cellular incorporation rate of Ac-LDL. N382Q/N393Q and N289Q/N382Q/N393Q were sequestered in the endoplasmic reticulum, resulting in a severe reduction in the cellular incorporation of Ac-LDL. N382Q showed a normal cell surface residency and an enhanced affinity for Ac-LDL, resulting in an elevated Ac-LDL cellular incorporation. These results indicate that the N-glycan of Asn(393) regulates the intracellular sorting of SREC-I and that the N-glycan of Asn(382) controls ligand-binding affinity. Furthermore, we detected an enhanced trypsin sensitivity of the N289Q. Glycan structure analyses revealed that the core-fucosylated bi-antennary is the common major structure at all glycosylation sites. In addition, tri- and tetra-antennary were detected as minor constituents at Asn(289). A bisecting GlcNAc was also detected at Asn(382) and Asn(393). Structural analyses and homology modeling of SREC-I suggest that the N-glycan bearing a β1-6GlcNAc branch at Asn(289) protects from proteinase attack and thus confers a higher stability on SREC-I. These data indicate that Asn(289)-, Asn(382)- and Asn(393)-linked N-glycans of SREC-I have distinct functions in regulating proteolytic resistance, ligand-binding affinity and subcellular localization, all of which might be involved in the development of atherogenesis.     

Counter-antigen presentation: fibroblasts produce cytokines by signalling through HLA class II molecules without inducing T-cell proliferation

Fibroblasts are known to express histocompatibility leukocyte antigen DR (HLA-DR) molecules on their cell surface upon stimulation with interferon gamma (IFN- gamma), while the exact roles of HLA-DR on fibroblasts remain undetermined. To understand the role of HLA-DR molecules on fibroblasts, we examined whether: (1) fibroblasts act as antigen presenting cells (APC) which activate helper T (Th) cells; and/or (2) fibroblasts are activated via HLA-II molecules by making a T-cell receptor (TCR)-peptide-major histocompatibility complex (MHC) complex. We used Th(0) clone HT8.3, which recognizes an antigenic peptide (Ag53 p141-161) in the context of DRB1*1501, as well as IFN - gamma - treated and irradiated periodontal ligament fibroblasts (PDL) expressing DRB1*1501 molecules. When peptide-pulsed fibroblasts were co-incubated with HT8.3 treated by the protein synthesis inhibitor emetine, peptide-induced de novo expression of lymphokines and cell-surface molecules on T cells can be neglected. The antigen presenting capacity of these fibroblasts was evaluated by examining the proliferative responses of Th cells. Possible activation of fibroblasts by stimulation via HLA-DR molecules was evaluated by quantitating secreted cytokines in the supernatants after 18-h culture with or without anti-HLA-DR monoclonal antibody (mAb) or emetine-treated HT8.3. Indeed, Th cells did not show proliferative responses when peptide-pulsed PDL were used as APC, whereas PDL produced larger amounts of interleukin (IL) 6, IL-8, monocyte chemoattractant protein 1 (MCP-1) and regulated upon activation, normal T expressed and secreted (RANTES) compared with controls, when cultured with anti-HLA-DR mAb or emetine-treated HT8.3. These findings suggest that HLA-DR expressed on fibroblasts do not present antigens to induce T-cell proliferation, but may act as receptor molecules that transmit signals into fibroblasts, based on DR-peptide-TCR interaction, resulting in the secretion of several cytokine species.     

Characterization of PomA mutants defective in the functional assembly of the Na(+)-driven flagellar motor in Vibrio alginolyticus

The polar flagellar motor of Vibrio alginolyticus rotates using Na(+) influx through the stator, which is composed of 2 subunits, PomA and PomB. About a dozen stators dynamically assemble around the rotor, depending on the Na(+) concentration in the surrounding environment. The motor torque is generated by the interaction between the cytoplasmic domain of PomA and the C-terminal region of FliG, a component of the rotor. We had shown previously that mutations of FliG affected the stator assembly around the rotor, which suggested that the PomA-FliG interaction is required for the assembly. In this study, we examined the effects of various mutations mainly in the cytoplasmic domain of PomA on that assembly. All mutant stators examined, which resulted in the loss of motor function, assembled at a lower level than did the wild-type PomA. A His tag pulldown assay showed that some mutations in PomA reduced the PomA-PomB interaction, but other mutations did not. Next, we examined the ion conductivity of the mutants using a mutant stator that lacks the plug domain, PomA/PomB(ΔL)(Δ41-120), which impairs cell growth by overproduction, presumably because a large amount of Na(+) is conducted into the cells. Some PomA mutations suppressed this growth inhibition, suggesting that such mutations reduce Na(+) conductivity, so that the stators could not assemble around the rotor. Only the mutation H136Y did not impair the stator formation and ion conductivity through the stator. We speculate that this particular mutation may affect the PomA-FliG interaction and prevent activation of the stator assembly around the rotor.     

Mutagenesis of the Residues Forming an Ion Binding Pocket of the NtpK Subunit of Enterococcus hirae V-ATPase

The crystal structures of the Na(+)- and Li(+)-bound NtpK rings of Enterococcus hirae V-ATPase have been obtained. The coupling ion (Na(+) or Li(+)) was surrounded by five oxygen atoms contributed by residues T64, Q65, Q110, E139, and L61, and the hydrogen bonds of the side chains of Q110, Y68, and T64 stabilized the position of the E139 γ carboxylate essential for ion occlusion (PDB accession numbers 2BL2 and 2CYD). We previously indicated that an NtpK mutant strain (E139D) lost tolerance to sodium but not to lithium at alkaline pHs and suggested that the E139 residue is indispensable for the enzymatic activity of E. hirae V-ATPase linked with the sodium tolerance of this bacterium. In this study, we examined the activities of V-ATPase in which these four residues, except for E139, were substituted. The V-ATPase activities of the Q65A and Y68A mutants were slightly retained, but those of the T64A and Q110A mutants were negligible. Among the residues, T64 and Q110 are indispensable for the ion coupling of E. hirae V-ATPase, in addition to the essential residue E139.     

Telomerase activity is not related to life history stage in the jellyfish Cassiopea sp.

The polyp (scyphistoma) of the jellyfish Cassiopea sp. can be maintained in culture for a long time, as polyps repeatedly reproduce asexually via formation of vegetative buds or propagules. The medusa, which is the sexually reproducing stage, typically has a relatively short life span. As a first step to understand the difference in life spans of the polyp and medusa stages of Cassiopea sp., we measured telomerase activity in different life cycle stages. We found telomerase activity in tissues of aposymbiotic polyps and propagules and symbiotic ephyrae (newly budded medusae) and adult medusae. No significant difference in telomerase activity was found between polyps and the bell region of the medusae. The cloned elongation products of the stretch PCR contained the TTAGGG repeats suggesting that the jellyfish has the ‘vertebrate’ telomere motif (TTAGGG)_n. This is the first study to show that somatic tissues of both polyp and medusa stages of a cnidarian had telomerase activity. Telomerase activity in somatic tissues may be related to the presence of multipotent interstitial cells and high regenerative capacity of cnidarians.     

Structure and organization of the rDNA intergenic spacer region in <Emphasis Type="Italic">Pythium ultimum</Emphasis>

Nucleotide sequences of the rDNA intergenic spacer (IGS) region in Pythium ultimum were determined in 16 clones obtained from three isolates differing in production of sexual organs. Several sequences with different lengths were detected in each isolate, showing heterogeneity in the IGS region. In addition, several tandem repeat regions were detected in all the clones. The sequences, length, and number of each copy largely varied among repeat regions. Length heterogeneity arose from the complex combination of the number of copy within the repeat regions. Furthermore, the nucleotide sequence of each copy and the number of repetition varied not only between isolates but also between clones from an isolate. Based on the sequence similarity and the number of copies in repeat regions, specific patterns different between homothallic P. ultimum and the Pythium group HS (hyphal swellings) were recognized in a few regions. These results suggest that these two groups have slight genetic differences in the IGS region, although the differences in most of the repeat regions were not enough to identify each group.     

Annual cycle of circulations in Lake Biwa, part 2: mechanisms

We investigate the mechanisms of circulations in Lake Biwa using model results that are driven by the momentum and heat fluxes evaluated from meteorological parameters observed around and on the lake. The kinetic energy budget shows that the wind stress over the lake, which has positive (negative) curl over the northern (southern) region of the lake throughout the year, generates the current system of multiple gyres in the stratified season (May–January), while the thermal forcing makes a minor contribution. This mechanism is quite different from the previous picture, in which the thermal forcing was dominant in forming the gyre system during the heating season. In the cooling season, the overturning of the lake occurs not in a simple one-dimensional (vertical) process, but in a time-dependent three-dimensional one. Cold water that is formed in the shallow eastern region descends the bottom slope to the deep lake in December to March while such a descent hardly occurs in the western region. A large shallow area (small heat capacity) as well as intense cooling in the eastern region effectively produces cold water. Wind again plays an important role by intensifying the cooling through its higher speed and by driving slope current. Further, the fluctuating wind direction causes significantly time-dependent behaviors of the slope current or bottom water formation.