Competent stripes for diverse positions of limbs/fins in gnathostome embryos

SUMMARY Every vertebrate species has its own unique morphology adapted to a particular lifestyle and habitat. Limbs and fins are strikingly diversified in size, shape, and position along the body axis. This diversity in morphology suggests the existence of a variety of embryonic developmental programs. However, comparisons of various embryos suggest common mechanisms underlying limb/fin formation. Here, we report the existence of continuous stripes of competency for appendage formation along the dorsal midline and the lateral trunk of all of the major jawed vertebrate (gnathostome) groups. We also show that the developing fin buds of cartilaginous fish share a mechanism of anterior–posterior axis formation as well as an shh (sonic hedgehog) expression domain in the posterior bud. We hypothesize a continuous distribution of competent stripes that represents the common developmental program at the root of appendage formation in gnathostomes. This schema would have permitted subsequent divergence into various levels of limbs/fins in each animal group.     

MG53 Regulates Membrane Budding and Exocytosis in Muscle Cells

Membrane recycling and remodeling contribute to multiple cellular functions, including cell fusion events during myogenesis. We have identified a tripartite motif (TRIM72) family member protein named MG53 and defined its role in mediating the dynamic process of membrane fusion and exocytosis in striated muscle. MG53 is a muscle-specific protein that contains a TRIM motif at the amino terminus and a SPRY motif at the carboxyl terminus. Live cell imaging of green fluorescent protein-MG53 fusion construct in cultured myoblasts showed that although MG53 contains no transmembrane segment it is tightly associated with intracellular vesicles and sarcolemmal membrane. RNA interference-mediated knockdown of MG53 expression impeded myoblast differentiation, whereas overexpression of MG53 enhanced vesicle trafficking to and budding from sarcolemmal membrane. Co-expression studies indicated that MG53 activity is regulated by a functional interaction with caveolin-3. Our data reveal a new function for TRIM family proteins in regulating membrane trafficking and fusion in striated muscles.When myoblasts exit the cell cycle during myogenesis, dramatic changes in membrane organization occur as myoblast fusion allows the formation of multinucleated muscle fibers. In addition to cell fusion events, differentiation of myotubes involves establishment of specialized membrane structures (1, 2). The transverse tubular invagination of sarcolemmal membrane and the intracellular membrane network known as the sarcoplasmic reticulum are two highly organized membrane architectures in cardiac and skeletal muscle. Establishment of these intricate membrane compartments requires extensive remodeling of the immature myoblast membranes. Dynamic membrane remodeling also contributes to many physiologic processes in mature muscle, including Ca²⁺ signaling, trafficking of glucose transporter (GLUT4), and other membrane internalization events involving caveolae structures (3-6). Although defects in membrane integrity have been linked to various forms of muscular dystrophy (7, 8), the molecular machinery regulating these specific membrane recycling and remodeling events in striated muscle is not well defined.The large tripartite motif (TRIM)⁵ family of proteins is involved in numerous cellular functions in a wide variety of cell types. Members of this protein family contain signature motifs that include a RING finger, a zinc binding moiety (B-box), and a coiled coil structure (RBCC), which invariably comprise the amino-terminal domain of TRIM family members (9). The carboxyl-terminal sequence of TRIM proteins is variable; in some cases a subfamily of TRIM proteins contains a SPRY domain, a sequence first observed in the ryanodine receptor Ca²⁺ channel in the sarcoplasmic reticulum membrane of excitable cells (10). Extensive studies have revealed that protein-protein interactions in the cytosol mediate the defined functions of TRIM proteins. For example, the ubiquitin E3 ligase enzymatic activity of several TRIM family members requires the B-box motif (11, 12). Recent studies have also indicated a role for TRIM proteins in defense against events involving membrane penetration, such as protection against infection by various viruses, including human immunodeficiency virus (13-15). Although most of the studies concentrate on the cytosolic action of TRIM, limited reports have investigated the role of TRIM proteins in membrane signaling or recycling.We have previously established an immunoproteomics approach that allows definition of novel components involved in myogenesis, Ca²⁺ signaling, and maintenance of membrane integrity in striated muscle (16). Using this approach, we have shown that junctophilin is a structural protein that establishes functional communication between sarcoplasmic reticulum and transverse tubule membranes at triad and dyad junctions in striated muscle (17-19). Further studies identified mitsugumin 29, a synaptophysin-related protein that is essential for biogenesis of triad membrane structures and Ca²⁺ signaling in skeletal muscle (20, 21). Screening of this immunoproteomics library led to the recent identification of MG53, a muscle-specific TRIM family protein (22). Domain homology analysis revealed that MG53 contains the prototypical RBCC motifs plus a SPRY domain at the carboxyl terminus. Genetic knock-out and functional studies reveal that MG53 nucleates the assembly of the sarcolemmal membrane repair machinery to restore cellular integrity following acute damage to the muscle fiber (22).Here we present evidence illustrating that MG53, in contrast to other known TRIM proteins, can localize to intracellular vesicles and the sarcolemmal membrane. A functional interaction between MG53 and caveolin-3, another muscle-specific protein, plays an essential role in regulating the dynamic process of membrane budding and exocytosis in skeletal muscle.     

Temperature dependence of proton permeation through a voltage-gated proton channel

Voltage-gated proton channels are found in many different types of cells, where they facilitate proton movement through the membrane. The mechanism of proton permeation through the channel is an issue of long-term interest, but it remains an open question. To address this issue, we examined the temperature dependence of proton permeation. Under whole cell recordings, rapid temperature changes within a few milliseconds were imposed. This method allowed for the measurement of current amplitudes immediately before and after a temperature jump, from which the ratios of these currents (I_ratio) were determined. The use of I_ratio for evaluating the temperature dependence minimized the contributions of factors other than permeation. Temperature jumps of various degrees (ΔT, −15 to 15°C) were applied over a wide temperature range (4–49°C), and the Q₁₀s for the proton currents were evaluated from the I_ratios. Q₁₀ exhibited a high temperature dependence, varying from 2.2 at 10°C to 1.3 at 40°C. This implies that processes with different temperature dependencies underlie the observed Q₁₀. A novel resistivity pulse method revealed that the access resistance with its low temperature dependence predominated in high temperature ranges. The measured temperature dependence of Q₁₀ was decomposed into Q₁₀ of the channel and of the access resistances. Finally, the Q₁₀ for proton permeation through the voltage-gated proton channel itself was calculated and found to vary from 2.8 at 5°C to 2.2 at 45°C, as expected for an activation enthalpy of 64 kJ/mol. The thermodynamic features for proton permeation through proton-selective channels were discussed for the underlying mechanism.     

Association of Thioautotrophic Bacteria with Deep-Sea Sponges

We investigated microorganisms associated with a deep-sea sponge, Characella sp. (Pachastrellidae) collected at a hydrothermal vent site (686 m depth) in the Sumisu Caldera, Ogasawara Island chain, Japan, and with two sponges, Pachastrella sp. (Pachastrellidae) and an unidentified Poecilosclerida sponge, collected at an oil seep (572 m depth) in the Gulf of Mexico, using polymerase chain reaction–denaturing gradient gel electrophoresis (PCR-DGGE) directed at bacterial 16S rRNA gene sequences. In the PCR-DGGE profiles, we detected a single clearly dominant band in each of the Characella sp. and the unidentified Poecilosclerida sponge. BLAST search of their sequences showed that they were most similar (>99% identity) to those of the gammaproteobacterial thioautotrophic symbionts of deep-sea bivalves from hydrothermal vents, Bathymodiolus spp. Phylogenetic analysis of the near-full length sequences of the 16S rRNA genes cloned from the unidentified Poecilosclerida sponge and Characella sp. confirmed that they were closely related to thioautotrophic symbionts. Although associations between sponges and methanotrophic bacteria have been reported previously, this is the first report of a possible stable association between sponges and thioautotrophic bacteria.     

Effect of laminaran accumulation on maturation in sporophyte fragments from Ecklonia cava (Phaeophyceae,Laminariales) under various laboratory light and temperature conditions

Fragments of Ecklonia cava Kjellman were cultured under controlled laboratory conditions of light irradiance, water temperature, and photoperiod. To clarify the relationship between the maturation of E. cava and the photosynthetic products, laminaran, the content in the fragments was measured with the progress of maturation. The culture conditions ranged from 12.5 to 100 µmol m^?2 s^?1, 10–25°C, and 14 : 10 h LD (light : dark) to 10 : 14 h LD. In the case of low light conditions, despite an optimum temperature for maturation, the fragments did not form sori and laminaran was not accumulated during the culture period. In the case of sufficient light and non‐optimum temperature conditions, the fragments did not form sori, but laminaran was accumulated. When the fragments were cultured under optimum light and temperature conditions for maturation, laminaran was accumulated in the early stage of maturation, just before or after cortex of the bladelets thickened, and decreased with the progress of maturation, and all fragments matured regardless of the length of the photoperiod. So, these results support the idea that laminaran is used as the main respiratory substrate in the maturation of E. cava.     

Production of wheat-Psathyrostachys huashanica chromosome addition lines

Psathyrostachys huashanica Keng (2n = 14; N(h)N(h)) is an endangered wheat-related species, with a distribution in the Huashan region of central China. It has many agronomically promising characters including resistance to disease and drought and winter hardiness. We produced hybrids between common wheat as the female parent and P. huashanica as the male parent. From the offspring, we selected chromosome addition lines of common wheat carrying each of all seven chromosomes of P. huashanica. Four chromosomes (B, D, E and F) were recovered in disomic lines and three (A, C and G) in monosomic addition lines. These alien chromosomes were distinguished from each other by cytological analyses. Chromosome A was characterized by a 45S rDNA site in the subtelomeric region of the short arm. Chromosome B carried one 5S and one 45S rDNA sites co-localized in an interstitial region of the short arm, and the expression of the alien high-molecular-weight glutenin was observed in the endosperm of line B. Chromosome D had a 45S rDNA signal in the interstitial region of the long arm. Chromosomes C, E, and F were distinguished by the EST-SSR markers Ltc0464, Ltc0096, and Xcfe175, respectively. The homoeologous group of each alien chromosome was implied from the results above, and the utilization of these addition lines for wheat breeding was discussed.     

Estimation of maturity of compost from food wastes and agro-residues by multiple regression analysis

The composting process of food wastes and tree cuttings was examined on four composting types composed from two kinds of systems and added mixture of microorganisms. The time courses of 32 parameters in each composting type were observed. The efficient composting system was found to be the static aerated reactor system in comparison with the turning pile one. Using the multiple regression analysis of all the data (159 samples) obtained from this study, some parameters were selected to predict the germination index (GI) value, which was adopted as a marker of compost maturity. For example, using the regression model generated from pH, NH(4)(+) concentration, acid phosphatase activity, and esterase activity of water extracts of the compost, GI value was expressed by the multi-linear regression equation (p<0.0001). High correlations between the measured GI value and the predicted one were made in each type of compost. As a result of these observations, the compost maturity might be predicted by only sensing of the water extract at the composting site without any requirements for a large-size equipment and skill, and this prediction system could contribute to the production of a stable compost in wide-spread use for the recycling market.     

Solution structure of the SWIRM domain of human histone demethylase LSD1

SWIRM is an evolutionarily conserved domain involved in several chromatin-modifying complexes. Recently, the LSD1 protein, which bears a SWIRM domain, was found to be a demethylase for Lys4-methylated histone H3. Here, we report a solution structure of the SWIRM domain of human LSD1. It forms a compact fold composed of 6 alpha helices, in which a 20 amino acid long helix (alpha4) is surrounded by 5 other short helices. The SWIRM domain structure could be divided into the N-terminal part (alpha1-alpha3) and the C-terminal part (alpha4-alpha6), which are connected to each other by a salt bridge. While the N-terminal part forms a SWIRM-specific structure, the C-terminal part adopts a helix-turn-helix (HTH)-related fold. We discuss a model in which the SWIRM domain acts as an anchor site for a histone tail.