Structural basis of sugar-recognizing ubiquitin ligase

SCF(Fbs1) is a ubiquitin ligase that functions in the endoplasmic reticulum (ER)-associated degradation pathway. Fbs1/Fbx2, a member of the F-box proteins, recognizes high-mannose oligosaccharides. Efficient binding to an N-glycan requires di-N-acetylchitobiose (chitobiose). Here we report the crystal structures of the sugar-binding domain (SBD) of Fbs1 alone and in complex with chitobiose. The SBD is composed of a ten-stranded antiparallel beta-sandwich. The structure of the SBD-chitobiose complex includes hydrogen bonds between Fbs1 and chitobiose and insertion of the methyl group of chitobiose into a small hydrophobic pocket of Fbs1. Moreover, NMR spectroscopy has demonstrated that the amino acid residues adjoining the chitobiose-binding site interact with the outer branches of the carbohydrate moiety. Considering that the innermost chitobiose moieties in N-glycans are usually involved in intramolecular interactions with the polypeptide moieties, we propose that Fbs1 interacts with the chitobiose in unfolded N-glycoprotein, pointing the protein moiety toward E2 for ubiquitination.     

Influence of photoperiod and temperature on reproductive mode in the Brine shrimp, Artemia franciscana

Brine shrimp, Artemia, exhibit two modes of reproduction: oviparity (diapause cyst production) and ovoviviparity (live larvae release). Environmental conditions determining these developmental routes are poorly understood, so we investigated the effects of photoperiod and temperature on reproductive mode. Nauplii of A. franciscana were hatched from cysts produced in the Great Salt Lake, Utah, and raised in 2% natural sea salt water under photoperiods of 24, 14, 12, or 10 h at 28 degrees or 20 degrees C. Mating pairs of mature shrimp were isolated and reared continuously under those conditions. The mode of reproduction shown by each pair was determined daily throughout their life span, and found to be greatly affected by photoperiod, and less influenced by temperature. The relative degree of oviparity increased as the photoperiod became shorter at both temperatures. In contrast, the degree of ovoviviparity was higher as the photoperiod became longer at both temperatures. The critical photoperiod appears to be between 12 and 14 h. For all photoperiods examined, the degree of oviparity was higher at 28 degrees C than at 20 degrees C, whereas the degree of ovoviviparity was greater at 20 degrees C than at 28 degrees C.     

Survival capacity of haploid-diploid goldfish chimeras

In teleosts, haploidy has been considered to be inviable due to the expression of abnormalities during embryogenesis, but the recent report of live haploid-diploid mosaic fish suggests the probable improvement of survival capacity by adding diploid cells or tissues to haploid embryos. In order to examine such possibilities, two types of haploid-diploid goldfish chimeric embryos were produced by transplantation of blastoderm between the normally fertilized diploid and the artificially induced gynogenetic haploid: the haploid-base chimera with the diploid upper half on the haploid lower half blastoderm and the diploid-base chimera with the haploid upper half on the diploid lower half blastoderm. Fluorescent detection of FITC-labeled cells, subsequent histochemical detection of biotin-labeled haploid cells and flow-cytometrical detection of both haploid and diploid cells proved successful induction of the haploid-diploid chimera. Both types of chimeric embryos demonstrated much better survival capacity than pure haploid individuals, but all the haploid-base chimeras died before 10 days after fertilization due to the expression of edema, whereas several diploid-base chimeras survived until 16 months after fertilization when the experiment was ended. This concluded diploid-base chimeras became viable by adding diploid cells to haploid embryos. However, the proportion of transplanted haploid cells was reduced and the distribution of these cells was limited to certain organs because survivors exhibited haploid cells only in brain, eye and/or skin. These results suggest possible elimination of haploid cells from the organs originated from ectoderm.     

Proteomics of the rice cell: systematic identification of the protein populations in subcellular compartments

Despite recent progress in sequencing the complete genome of rice (Oryza sativa), the proteome of this species remains poorly understood. To extend our knowledge of the rice proteome, the subcellular compartments, which include plasma membranes (PM), vacuolar membranes (VM), Golgi membranes (GM), mitochondria (MT), and chloroplasts (CP), were purified from rice seedlings and cultured suspension cells. The proteins of each of these compartments were then systematically analyzed using two-dimensional (2D) electrophoresis, mass spectrometry, and Edman sequencing, followed by database searching. In all, 58 of the 464 spots detected by 2D electrophoresis in PM, 43 of the 141 spots in VM, 46 of the 361 spots in GM, 146 in the 672 spots in MT, and 89 of the 252 spots in CP could be identified by this procedure. The characterized proteins were found to be involved in various processes, such as respiration and the citric acid cycle in MT; photosynthesis and ATP synthesis in CP; and antifungal defense and signal systems in the membranes. Edman degradation revealed that 60–98% of N-terminal sequences were blocked, and the ratios of blocked to unblocked proteins in the proteomes of the various subcellular compartments differed. The data on the proteomes of subcellular compartments in rice will be valuable for resolving questions in functional genomics as well as for genome-wide exploration of plant function.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by G. Jürgens     

The initial stages of oogenesis and their relation to differential fertility in the honey bee (Apis mellifera) castes

Neither the overall differences in ovariole number nor the caste-specifically modulated expression of vitellogenin can fully explain the striking caste differences in honey bee reproduction, in particular the mechanisms that block oogenesis in virgin queens and in workers kept in the presence of a queen. For this reason we investigated the initial stages of oogenesis in queens in relation to mating status and in workers exposed to different social conditions. A striking feature in ovarioles of both castes was a considerably elongated terminal filament which consisted not only of normal terminal filament cells but also contained apparently undifferentiated cells that were tentatively considered as stem cells. BrdU incorporation was detected in the upper germarium, as well as in the terminal filament. Cytoskeleton analysis by TRITC-phalloidin labeling for F-actin, and immunofluorescence detection for β-tubulin did not reveal structural differences in the early oogenesis steps between queens and queenless workers. In contrast, queenright workers showed signs of a disorganized microtubule and microfilament system that could explain the histological evidence for progressive cell death observed in the germaria. In addition to cytoplasmic tubulin we also detected marked intranuclear foci indicating the presence of nuclear β_II-tubulin.     

Improved synthesis with high yield and increased molecular weight of poly(alpha,beta-malic acid) by direct polycondensation

The development of synthetic biodegradable polymers, such as poly(alpha-hydroxy acid), is particularly important for constructing medical devices, including scaffolds and sutures, and has attracted growing interest in the biomedical field. Here, we report a novel approach to preparing high molecular weight poly(malic acid) (HMW--PMA) as a biodegradable and bioabsorbable water-soluble polymer. We investigated in detail the reaction conditions for the simple direct polycondensation of l-malic acid, including the reaction times, temperatures, and catalysts. The molecular weight of synthesized alpha,beta-PMA is dependent on both the reaction temperature and time. The optimum reaction condition to obtain alpha,beta-PMA by direct polycondensation using tin(II) chloride as a catalyst was thus determined to be 110 degrees C for 45 h with a molecular weight of 5300. The method for alpha,beta-PMA synthesis established here will facilitate production of alpha,beta-PMA of various molecular weights, which may have a potential utility as biomaterials.     

Adhesion behavior of peritoneal cells on the surface of self-assembled triblock copolymer hydrogels

Adhesion behavior of cells to the surface of physical hydrogel membranes prepared by water-induced self-organization of precisely synthesized ABA-triblock copolymers comprised of poly(beta-benzyl L-aspartate) (PBLA) as A segment and poly(ethylene oxide) (PEO, molecular weight = 20 000) as the B segment were investigated. The cast film from the methylenechloride solution of these copolymers swelled in water very rapidly forming hydrogels (100-400% water content of total weight). The content of PBLA affected the strength, the hydrophobicity, and the amount of water involved in the hydrogel surface. During the early stage of cultivation with murine peritoneal cells, cell adhesion on the hydrogels of PEO and PBLA with 18 (20K18) and 25 (20K25) monomeric units was not observed, while adhesion on the hydrogels of PEO and PBLA with 32 (20K32) and 55 (20K55) monomeric units was successful, suggesting more than 12 mol % in PBLA content is necessary for adhesion of these cells. Although cell spreading on the hydrogels of 20K18, 20K25, and 20K32 was not sufficient, the hydrogel of 20K55 allowed cell adhesion and spreading to be bipolar with leading edge whose raffling is active with pseudopodium and lamellipodium as well as PBLA homopolymer, suggesting active motility of these cells. Remarkably, prolonged incubation restored adhesiveness onto the films at 20K18 in contrast to adhesion with 20K25 despite low hydrophobicity. It is conceivable that adaptation of proteins and chemical changes to the surface during the culture period may participate in these phenomena. Mechanical properties and interaction between cell and these copolymer hydrogels could be controlled by composition of block segments, and optimization for implants could also be attainable.     

Dorsal telencephalon-specific expression of Cre recombinase in PAC transgenic mice

The ability to restrict gene expression or disruption to specific regions of the brain would enhance understanding of the molecular basis for brain development and function. For this purpose, brain region-restricted promoters are essential. Here we report the isolation of a DNA fragment containing the Emx1 gene promoter, which is responsible for dorsal telencephalon-specific expression. The Cre recombinase gene was inserted into a mouse PAC (P1-derived artificial chromosome) Emx1-locus clone (PAC-Emx1#1 clone) and utilized to generate three transgenic mouse lines. In all three lines, especially Tg3, Cre-mediated recombination was highly restricted to Emx1-expressing cell lineages, from embryonic stages to adulthood. Immunohistochemical analyses showed that Cre protein is expressed in the dorsal telencephalon in all three lines in adulthood. Thus, the PAC-Emx1#1 clone contains essentially all regulatory elements necessary for Emx1 gene expression. Our results suggest that Emx1-Cre Tg3 mice and the PAC-Emx1#1 clone constitute powerful tools for dorsal telencephalon-specific gene manipulation.     

Solution structure of the RWD domain of the mouse GCN2 protein

GCN2 is the alpha-subunit of the only translation initiation factor (eIF2alpha) kinase that appears in all eukaryotes. Its function requires an interaction with GCN1 via the domain at its N-terminus, which is termed the RWD domain after three major RWD-containing proteins: RING finger-containing proteins, WD-repeat-containing proteins, and yeast DEAD (DEXD)-like helicases. In this study, we determined the solution structure of the mouse GCN2 RWD domain using NMR spectroscopy. The structure forms an alpha + beta sandwich fold consisting of two layers: a four-stranded antiparallel beta-sheet, and three side-by-side alpha-helices, with an alphabetabetabetabetaalphaalpha topology. A characteristic YPXXXP motif, which always occurs in RWD domains, forms a stable loop including three consecutive beta-turns that overlap with each other by two residues (triple beta-turn). As putative binding sites with GCN1, a structure-based alignment allowed the identification of several surface residues in alpha-helix 3 that are characteristic of the GCN2 RWD domains. Despite the apparent absence of sequence similarity, the RWD structure significantly resembles that of ubiquitin-conjugating enzymes (E2s), with most of the structural differences in the region connecting beta-strand 4 and alpha-helix 3. The structural architecture, including the triple beta-turn, is fundamentally common among various RWD domains and E2s, but most of the surface residues on the structure vary. Thus, it appears that the RWD domain is a novel structural domain for protein-binding that plays specific roles in individual RWD-containing proteins.