Genetic population structure of a reef goby, <Emphasis Type="Italic">Bathygobius cocosensis</Emphasis>, in the northwestern Pacific

To investigate the genetic population structure of a reef goby, Bathygobius cocosensis, in the northwestern Pacific in relation to major oceanic currents (North Equatorial and Kuroshio Currents), the mitochondrial DNA (mtDNA) phylogeny and demography of the species were inferred from partial nucleotide sequences of the mitochondrial NADH dehydrogenase 5 subunit (ND5) gene (945 bp). The resultant mtDNA tree and population genetic analyses showed that the Bonin Islands’ population apparently differed from the other populations in the Japan–Ryukyu–Guam region, which lie along the routes of the North Equatorial and Kuroshio Currents. A test calculation indicated the divergence date to be approximately 1 million years ago. The population expansion parameters indicated that the Bonin Islands’ population had survived during the fluctuations in sea level in the Pleistocene–Holocene age and that the population had expanded thereafter.     

Non-core region modulates interleukin-11 signaling activity: generation of agonist and antagonist variants

Human interleukin-11 (hIL-11) is a pleiotropic cytokine administered to patients with low platelet counts. From a structural point of view hIL-11 belongs to the long-helix cytokine superfamily, which is characterized by a conserved core motif consisting of four α-helices. We have investigated the region of hIL-11 that does not belong to the α-helical bundle motif, and that for the purpose of brevity we have termed "non-core region." The primary sequence of the interleukin was altered at various locations within the non-core region by introducing glycosylation sites. Functional consequences of these modifications were examined in cell-based as well as biophysical assays. Overall, the data indicated that the non-core region modulates the function of hIL-11 in two ways. First, the majority of muteins displayed enhanced cell-stimulatory properties (superagonist behavior) in a glycosylation-dependent manner, suggesting that the non-core region is biologically designed to limit the full potential of hIL-11. Second, specific modification of a predicted mini α-helix led to cytokine inactivation, demonstrating that this putative structural element belongs to site III engaging a second copy of cell-receptor gp130. These findings have unveiled new and unexpected elements modulating the biological activity of hIL-11, which may be exploited to develop more versatile medications based on this important cytokine.     

Biochemical studies on sphingolipids of Artemia franciscana: novel neutral glycosphingolipids

Neutral glycosphingolipids containing one to six sugars in their oligosaccharide chains have been isolated from cysts of the brine shrimp Artemia franciscana. The structures of these glycolipids were identified by methylation analysis, partial acid hydrolysis, gas-liquid chromatography, combined gas-liquid chromatography-mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and proton nuclear magnetic resonance spectroscopy to be Glcβ1-Cer, Manβ1-4Glcβ1-Cer, Fucα1-3Manβ1-4Glcβ1-Cer, GlcNAcβ1-3Manβ1-4Glcβ1-Cer, GlcNAcα1-2Fucα1-3Manβ1-4Glcβ1-Cer, GalNAcβ1-4GlcNAcβ1-3Manβ1-4Glcβ1-Cer, GalNAcβ1-4(Fucα1-3)GlcNAcβ1-3Manβ1-4Glcβ1-Cer (CPS), and GalNAcβ1-4(GlcNAcα1-2Fucα1-3)GlcNAcβ1-3Manβ1-4Glcβ1-Cer (CHS). Two glycosphingolipids, CPS and CHS, were characterized as novel structures. Because Artemia contains a certain series of glycosphingolipids (-Fucα3Manβ4GlcβCer), which differ from the core sugar sequences reported thus far, we tentatively designated the glycosphingolipids characterized as nonarthro-series ones. Furthermore, CHS exhibited a hybrid structure of arthro-series and nonarthro-series sugar chain. Two novel glycosphingolipids were characterized from the brine shrimp Artemia franciscana; one was composed of arthrotetraose and a branching fucose attached to N-acetylglucosamine residue, and the other was composed of CPS with an additional N-acetylglucosamine residue attached to the branching fucose.     

Ameloblastin regulates osteogenic differentiation by inhibiting Src kinase via cross talk between integrin beta1 and CD63

Ameloblastin, the most abundant nonamelogenin enamel matrix protein, plays a role in ameloblast differentiation. Here, we found that ameloblastin was expressed in osteosarcoma cells; to explore the potential functions of ameloblastin in osteoblasts, we investigated whether this protein is involved in osteogenic differentiation and bone formation on the premise that CD63, a member of the transmembrane-4 glycoprotein superfamily, interacts with integrins in the presence of ameloblastin. Ameloblastin bound to CD63 and promoted CD63 binding to integrin β1. The interaction between CD63 and integrin β1 induced Src kinase inactivation via the binding of CD63 to Src. The reduction of Src activity and osteogenic differentiation mediated by ameloblastin were abrogated by treatment with anti-CD63 antibody and overexpression of constitutively active Src, respectively. Therefore, our results suggest that ameloblastin is expressed in osteoblasts and functions as a promoting factor for osteogenic differentiation via a novel pathway through the interaction between CD63 and integrin β1.     

A New Class of Endoplasmic Reticulum Export Signal ΦXΦXΦ for Transmembrane Proteins and Its Selective Interaction with Sec24C

Protein export from the endoplasmic reticulum (ER) depends on the interaction between a signal motif on the cargo and a cargo recognition site on the coatomer protein complex II. A hydrophobic sequence in the N terminus of the bovine anion exchanger 1 (AE1) anion exchanger facilitated the ER export of human AE1Δ11, an ER-retained AE1 mutant, through interaction with a specific Sec24 isoform. The cell surface expression and N-glycan processing of various substitution mutants or chimeras of human and bovine AE1 proteins and their Δ11 mutants in HEK293 cells were examined. The N-terminal sequence (V/L/F)X(I/L)X(M/L), ²⁶VSIPM³⁰ in bovine AE1, which is comparable with ΦXΦXΦ, acted as the ER export signal for AE1 and AE1Δ11 (Φ is a hydrophobic amino acid, and X is any amino acid). The AE1-Ly49E chimeric protein possessing the ΦXΦXΦ motif exhibited effective cell surface expression and N-glycan maturation via the coatomer protein complex II pathway, whereas a chimera lacking this motif was retained in the ER. A synthetic polypeptide containing the N terminus of bovine AE1 bound the Sec23A-Sec24C complex through a selective interaction with Sec24C. Co-transfection of Sec24C-AAA, in which the residues ⁸⁹⁵LIL⁸⁹⁷ (the binding site for another ER export signal motif IXM on Sec24C and Sec24D) were mutated to ⁸⁹⁵AAA⁸⁹⁷, specifically increased ER retention of the AE1-Ly49E chimera. These findings demonstrate that the ΦXΦXΦ sequence functions as a novel signal motif for the ER export of cargo proteins through an exclusive interaction with Sec24C.     

Construction of a Fibrobacter succinogenes Genomic Map and Demonstration of Diversity at the Genomic Level

The genomic cleavage map of the type strain Fibrobacter succinogenes S85 was constructed. The restriction enzymes AscI, AvrII, FseI, NotI, and SfiI generated DNA fragments of suitable size distribution that could be resolved by pulsed-field gel electrophoresis (PFGE). An average genome size of 3.6 Mb was obtained by summing the total fragment sizes. The linkages between the 15 AscI fragments of the genome were determined by combining two approaches: isolation of linking clones and cross-hybridization of restriction fragments. The genome of F. succinogenes was found to be represented by the single circular DNA molecule. Southern hybridization with specific probes allowed the eight genetic markers to be located on the restriction map. The genome of this bacterium contains at least three rRNA operons. PFGE of the other three strains of F. succinogenes gave estimated genome sizes close to that of the type strain. However, RFLP patterns of these strains generated by AscI digestion are completely different. Pairwise comparison of the genomic fragment distribution between the type strain and the three isolates showed a similarity level in the region of 14.3% to 31.3%. No fragment common to all of these F. succinogenes strains could be detected by PFGE. A marked degree of genomic heterogeneity among members of this species makes genomic RFLP a highly discriminatory and useful molecular typing tool for population studies. Received: 23 October 1996 / Accepted: 31 December 1996