Plasmatocyte spreading peptide (PSP1) and growth blocking peptide (GBP) are multifunctional homologs

Recently, we identified Plasmatocyte spreading peptide (PSP1) from the moth Pseudoplusia includens and reported that it mediates adhesion of hemocytes to foreign surfaces. PSP1 is structurally very similar to three classes of peptides identified earlier from other species of Lepidoptera: growth blocking peptide (GBP) originally identified in Pseudaletia separata, and a series of related peptides from other species designated as paralytic (PP) or cardioactive (CAP) peptides. In this study, we conducted parallel experiments in P. includens and P. separata to determine whether PSP1 and GBP have distinct or multiple biological activities. Both peptides affected the adhesive state of hemocytes from each moth very similarly. PSP1 and GBP exhibited significant growth blocking and paralytic activity in P. separata. Both peptides also had growth blocking activity in P. includens although larvae had to be injected with higher doses of each peptide to reduce weight gain than was observed for P. separata. However, GBP and PSP1 had little paralytic activity in P. includens. Collectively, our results indicate that GBP and PSP1 are multifunctional, but that some interspecific variation also exists in their growth blocking and paralytic activities. We suggest that all PSP1, GBP, PP and CAP family members are homologs that likely have multiple biological activities. Based upon the unique consensus sequence of their N termini, we propose that these molecules be henceforth referred to as members of the "ENF" peptide family.     

Orientation of Smooth Muscle-Derived A10 Cells in Culture by Cyclic Stretching: Relationship between Stress Fiber Rearrangement and Cell Reorientation

Mechanical stress causes various responses in cells both in vivo and in vitro. Realignment of cells and stress fibers is one of the remarkable phenomena that are induced by the stress. However, the mechanism by which their realignment is controlled is largely unknown. In this study, effects of mechanical stretch on the morphology of cultured cells were examined using a cyclic and reciprocal cell stretching apparatus. A10 cells, a cell line derived from rat aortic smooth muscle, were used as a model, since they are spindle-shaped and have remarkable stress fibers aligned along the longitudinal cell axis. Therefore, the orientation of the cell and stress fibers could be easily identified. When the cells were cultured on elastic silicone membranes and subjected to cyclic and reciprocal stretch with an amplitude of 20% at a frequency of 60 cycles per minute, actin stress fibers were aligned obliquely to the direction of stretching with angles of 50 to 70 degrees within about 15 min after the onset of stretching. Then, after 1-3 hr of cyclic stretching, the long axes of a majority of the cells were also reoriented to similar directions to the stress fibers. The stretch-induced cell reorientation was blocked by 1 muM cytochalasin B, but not by colcemid. These results indicate that the orientation of cells and actin filaments are closely related and actin filaments play a critical role in the early step of the cell reorientation.     

Organ and cellular localization of asparagine synthetase in rice plants

DNA gel blot analysis suggested that asparagine synthetase (AS; EC 6.3.5.4) occurred as a single gene in rice. A fusion protein consisting of 17 kDa tagged-region from pET32a(+) expression plasmid and 42 kDa N-terminal region of rice AS was first expressed in Escherichia coli. The resulting polypeptide was purified and a mono-specific antibody for rice AS was prepared after affinity-purification with the antigen. Immunoblotting revealed a high content of AS protein in the leaf sheath at the second position from the fully expanded top leaf and in grains at the middle stage of ripening. Accumulation of mRNA for AS was also observed in these organs. During the ripening of the spikelets, the AS protein contents increased during the first 21 days after flowering, then declined rapidly. Immunolocalization analysis revealed signals for AS protein in the companion cells of vascular bundles of leaf sheath and phloem-parenchyma cells, nucellar projection, and nucellar epidermis of dorsal vascular bundles of grains.     

Genetic linkage of Bietti crystallin corneoretinal dystrophy to chromosome 4q35

Bietti crystalline corneoretinal dystrophy (BCD) is an autosomal recessive retinal degeneration characterized by multiple glistening intraretinal dots scattered over the fundus, degeneration of the retina, and sclerosis of the choroidal vessels, ultimately resulting in progressive night blindness and constriction of the visual field. Although BCD has been associated with abnormalities in fatty-acid metabolism and absence of fatty-acid binding by two cytosolic proteins, the genetic basis of BCD is unknown. We report linkage of the BCD locus to D4S426 (maximum LOD score [Z(max)] 4.81; recombination fraction [straight theta] 0), D4S2688 (Zmax=3.97; straight theta=0), and D4S2299 (Zmax=5.31; straight theta=0), on chromosome 4q35-4qtel. Multipoint analysis confirmed linkage to the region telomeric of D4S1652 with a Z(max) of 5.3 located 4 cM telomeric of marker D4S2930.     

Crystal structure of Pseudomonas aeruginosa PAK pilin suggests a main-chain-dominated mode of receptor binding

Fibers of pilin monomers (pili) form the dominant adhesin of Pseudomonas aeruginosa, and they play an important role in infections by this opportunistic bacterial pathogen. Blocking adhesion is therefore a target for vaccine development. The receptor-binding site is located in a C-terminal disulphide-bonded loop of each pilin monomer, but functional binding sites are displayed only at the tip of the pilus. A factor complicating vaccination is that different bacterial strains produce distinct, and sometimes highly divergent, pilin variants. It is surprising that all strains still appear to bind a common receptor, asialo-GM1. Here, we present the 1.63 A crystal structure of pilin from P. aeruginosa strain PAK. The structure shows that the proposed receptor-binding site is formed by two beta-turns that create a surface dominated by main-chain atoms. Receptor specificity could therefore be maintained, whilst allowing side-chain variation, if the main-chain conformation is conserved. The location of the binding site relative to the proposed packing of the pilus fiber raises new issues and suggests that the current fiber model may have to be reconsidered. Finally, the structure of the C-terminal disulphide-bonded loop will provide the template for the structure-based design of a consensus sequence vaccine.     

Interferon Yield and MHC Antigen Expression of Human Medulloblastoma Cells and Its Suppression During Dibutyryl Cyclic AMP-Induced Differentiation: Do Medulloblastoma Cells Derive from Bipotent Neuronal and Glial Progenitors?

1. Human medulloblastoma (ONS-76), a central nervous system (CNS)-derived undifferentiated cell line, was found to possess glial characteristics as defined by responses in the interferon (IFN) system; ONS-76 cells produced as much IFN- as human fibroblast and glioma cells by viral infection and poly(I):poly(C) induction.2. Major histocompatibility complex (MHC) class I antigens were also induced under IFN- stimulation. ONS-76 cells expressed neurofilament protein, as shown by Northern blot analysis, and morphological differentiation was induced by dibutyryl cyclic AMP (dcAMP).3. Expression of IFN- and MHC class I antigens was suppressed in ONS-76 cells during the dcAMP-induced differentiation.4. These results showed that ONS-76 cells possessed a glial properly in IFN system responses and a neuronal property in cytoskeleton protein, suggesting that the precursors of medulloblastoma may be characterized as bipotent neuronal and glial progenitors in CNS.     

Function and Evolutionary Origin of Unicellular Camera-Type Eye Structure

The ocelloid is an extraordinary eyespot organelle found only in the dinoflagellate family Warnowiaceae. It contains retina- and lens-like structures called the retinal body and the hyalosome. The ocelloid has been an evolutionary enigma because of its remarkable resemblance to the multicellular camera-type eye. To determine if the ocelloid is functionally photoreceptive, we investigated the warnowiid dinoflagellate Erythropsidinium. Here, we show that the morphology of the retinal body changed depending on different illumination conditions and the hyalosome manifests the refractile nature. Identifying a rhodopsin gene fragment in Erythropsidinium ESTs that is expressed in the retinal body by in situ hybridization, we also show that ocelloids are actually light sensitive photoreceptors. The rhodopsin gene identified is most closely related to bacterial rhodopsins. Taken together, we suggest that the ocelloid is an intracellular camera-type eye, which might be originated from endosymbiotic origin.     

Stimulation of Macrophages with the β-Glucan Produced by Aureobasidium pullulans Promotes the Secretion of Tumor Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL)

A β-glucan produced by Aureobasidium pullulans (AP-PG) is consisting of a β-(1,3)-linked main chain with β-(1,6)-linked glucose side residues. Various β-glucans consisting of β-(1,3)-linked main chain including AP-PG are believed to exhibit anti-tumor activities, and actually, anti-tumor activities of AP-PG in mice have been demonstrated. In this study, we demonstrate that stimulation with AP-PG induces TRAIL expression in mouse and human macrophage-like cell lines. TRAIL is known to be a cytokine which specifically induces apoptosis in transformed cells, but not in untransformed cells. The expression of TRAIL mRNA after stimulation with AP-PG was increased in RAW264.7 cells, Mono Mac 6 cells, and macrophage-differentiated THP-1 cells. The mRNA expression of TNF-α and FasL is only weakly increased after stimulation with AP-PG. The induction activity of TRAIL by curdlan, a bacterial β-glucan, was very similar to that by AP-PG in RAW264.7 cells, but weaker in macrophage-differentiated THP-1 cells. Activation of caspases was found in HeLa cells after treatment with the supernatant of cultured medium from AP-PG-stimulated Mono Mac 6 cells, and was inhibited by the anti-TRAIL neutralizing antibody. These findings suggest that the stimulation with AP-PG effectively induces TRAIL in macrophages, and that it may be related to apoptosis induction of tumor cells.     

Non-synonymous FGD3 Variant as Positional Candidate for Disproportional Tall Stature Accounting for a Carcass Weight QTL (CW-3) and Skeletal Dysplasia in Japanese Black Cattle

Recessive skeletal dysplasia, characterized by joint- and/or hip bone-enlargement, was mapped within the critical region for a major quantitative trait locus (QTL) influencing carcass weight; previously named CW-3 in Japanese Black cattle. The risk allele was on the same chromosome as the Q allele that increases carcass weight. Phenotypic characterization revealed that the risk allele causes disproportional tall stature and bone size that increases carcass weight in heterozygous individuals but causes disproportionately narrow chest width in homozygotes. A non-synonymous variant of FGD3 was identified as a positional candidate quantitative trait nucleotide (QTN) and the corresponding mutant protein showed reduced activity as a guanine nucleotide exchange factor for Cdc42. FGD3 is expressed in the growth plate cartilage of femurs from bovine and mouse. Thus, loss of FDG3 activity may lead to subsequent loss of Cdc42 function. This would be consistent with the columnar disorganization of proliferating chondrocytes in chondrocyte-specific inactivated Cdc42 mutant mice. This is the first report showing association of FGD3 with skeletal dysplasia.     

Bacteria Endosymbiont,Wolbachia, Promotes Parasitism of Parasitoid Wasp Asobara japonica

Wolbachia is the most widespread endosymbiotic bacterium that manipulates reproduction of its arthropod hosts to enhance its own spread throughout host populations. Infection with Wolbachia causes complete parthenogenetic reproduction in many Hymenoptera, producing only female offspring. The mechanism of such reproductive manipulation by Wolbachia has been extensively studied. However, the effects of Wolbachia symbiosis on behavioral traits of the hosts are scarcely investigated. The parasitoid wasp Asobara japonica is an ideal insect to investigate this because symbiotic and aposymbiotic strains are available: Wolbachia-infected Tokyo (TK) and noninfected Iriomote (IR) strains originally collected on the main island and southwest islands of Japan, respectively. We compared the oviposition behaviors of the two strains and found that TK strain females parasitized Drosophila melanogaster larvae more actively than the IR strain, especially during the first two days after eclosion. Removing Wolbachia from the TK strain wasps by treatment with tetracycline or rifampicin decreased their parasitism activity to the level of the IR strain. Morphological and behavioral analyses of both strain wasps showed that Wolbachia endosymbionts do not affect development of the host female reproductive tract and eggs, but do enhance host-searching ability of female wasps. These results suggest the possibility that Wolbachia endosymbionts may promote their diffusion and persistence in the host A. japonica population not only at least partly by parthenogenesis but also by enhancement of oviposition frequency of the host females.