Defective smooth muscle development in qkI-deficient mice

The qkI gene encodes an RNA binding protein which was identified as a candidate for the classical neurologic mutation, qkv. Although qkI is involved in glial cell differentiation in mice, qkI homologues in other species play important roles in various developmental processes. Here, we show a novel function of qkI in smooth muscle cell differentiation during embryonic blood vessel formation. qkI null embryos died between embryonic day 9.5 and 10.5. Embryonic day 9.5 qkI null embryos showed a lack of large vitelline vessels in the yolk sacs, kinky neural tubes, pericardial effusion, open neural tubes and incomplete embryonic turning. Using X-gal and immunohistochemical staining, qkI is first shown to be expressed in endothelial cells and smooth muscle cells. Analyses of qkI null embryos in vivo and in vitro revealed that the vitelline artery was too thin to connect properly to the yolk sac, thereby preventing remodeling of the yolk sac vasculature, and that the vitelline vessel was deficient in smooth muscle cells. Addition of QKI and platelet-endothelial cell adhesion molecule-1 positive cells to an in vitro para-aortic splanchnopleural culture of qkI null embryos rescued the vascular remodeling deficit. These data suggest that QKI protein has a critical regulatory role in smooth muscle cell development, and that smooth muscle cells play an important role in inducing vascular remodeling.     

PKCdelta inhibits PKCalpha-mediated activation of phospholipase D1 in a manner independent of its protein kinase activity

The regulation of phospholipase D1 (PLD1) by protein kinase C (PKC) isoforms was analyzed in human melanoma cell lines. 12-O-Tetradecanoylphorbol-13-acetate (TPA)-induced PLD1 activation was suppressed by the introduction of PKCdelta as well as its kinase-negative mutant in MeWo cells, which contain PKCalpha but lack PKCbeta. PLD activity was not affected by PKCdelta in G361 cells, which have PKCbeta but are deficient in PKCalpha. In MeWo cells introduced by PKCalpha and PLD1, the association of these proteins was observed, which was enhanced by the TPA treatment. In cells overexpressing PKCdelta in addition to PKCalpha and PLD1, TPA treatment increased the association of PKCdelta and PLD1, while it attenuated the association of PKCalpha and PLD1. These results indicate that PKCdelta inhibits TPA-induced PLD1 activation mediated by PKCalpha through the association with PLD1.     

Negamycin restores dystrophin expression in skeletal and cardiac muscles of mdx mice

The ability of aminoglycoside antibiotics to promote read-through of nonsense mutations has attracted interest in these drugs as potential therapeutic agents in genetic diseases. However, the toxicity of aminoglycoside antibiotics may result in severe side effects during long-term treatment. In this paper, we report that negamycin, a dipeptide antibiotic, also restores dystrophin expression in skeletal and cardiac muscles of the mdx mouse, an animal model of Duchenne muscular dystrophy (DMD) with a nonsense mutation in the dystrophin gene, and in cultured mdx myotubes. Dystrophin expression was confirmed by immunohistochemistry and immunoblotting. We also compared the toxicity of negamycin and gentamicin, and found negamycin to be less toxic. Furthermore, we demonstrate that negamycin binds to a partial sequence of the eukaryotic rRNA-decoding A-site. We conclude that negamycin is a promising new therapeutic candidate for DMD and other genetic diseases caused by nonsense mutations.     

Mating and reproduction of predaceous diving beetles,Dytiscus sharpi,observed under artificial breeding conditions

Mating season and embryonic development of the predaceous diving beetles, Dytiscus sharpi, (Coleoptera; Dytiscidae) were observed under artificial breeding conditions. Female and male adult insects started mating from November to March and gave first instar larvae mainly in April. When the mating was artificially delayed until February, first instar larvae appeared from the end of March to the middle of May. I also investigated the effects of temperature on larval development. Apparent hatchability of eggs was not affected by high temperature, however, their normal development after hatching was significantly interfered. Most of the first instar larvae kept at 20-25 degrees C from before hatching died within one day after hatching. By contrast, juveniles kept outdoors (7.0-20.9 degrees C) could develop at least until second instar larvae. Temperature >23 degrees C after hatching had no effects on larval development. From these observations, it was concluded that the reproduction strategy of Dytiscus sharpi, i.e. mating in late autumn and hatching in early spring would be the reasonable results of adaptation to the warm habitats where they are collected.     

Sex identification by alternative polymerase chain reaction methods in falconiformes

A number of avian species are difficult to sex morphologically, especially as nestlings. Like other avian species, many species of Falconiformes are sexually monomorphic. Therefore, it is desirable that new methods based on DNA analysis are established in Falconiformes and other sexual monomorphic species. We identified sex in Falconiformes by two alternative methods. First, we used a sexing method based on the intronic length variation between CHD1W and CHD1Z using primers flanking the intron. In this method, two species of Falconidae could be identified for sexing. However, six species of Accipitridae could not, because they have few length variations. The second method used was based on differences in sequences between CHD1W and CHD1Z. From sequence analysis, a 3'-terminal mismatch primer on point mutation conserved among Falconiformes was designed, and identification of sex with the amplification refractory mutation system (ARMS) was performed. This method could identify sex in all species tested. In addition, because the 3'-terminal mismatch primer was designed on a point mutation conserved among Falconiformes, ARMS with these primers may identify sex in all Falconiformes. These are simple and rapid sexing methods, since only polymerase chain reaction (PCR) and agarose electrophoresis are required. In conclusion, sex identification by an alternative PCR approach based on intronic length variation and on differences in sequences between CHD1W and CHD1Z proved applicable to and useful for Falconiformes.     

Structural elements in the internal ribosome entry site of Plautia stali intestine virus responsible for binding with ribosomes

Plautia stali intestine virus (PSIV) has an internal ribosome entry site (IRES) at the intergenic region of the genome. The PSIV IRES initiates translation with glutamine rather than the universal methionine. To analyze the mechanism of IRES-mediated initiation, binding of IRES RNA to salt-washed ribosomes in the absence of translation factors was studied. Among the three pseudoknots (PKs I, II and III) within the IRES, PK III was the most important for ribosome binding. Chemical footprint analyses showed that the loop parts of the two stem–loop structures in Domain 2, which are highly conserved in related viruses, are protected by 40S but not by 60S ribosomes. Because PK III is close to the two loops, these structural elements were considered to be important for binding of the 40S subunit. Competitive binding analyses showed that the IRES RNA does not bind poly(U)-programmed ribosomes preincubated with tRNA^Phe or its anticodon stem– loop (ASL) fragment. However, Domain 3-deleted IRES bound to programmed ribosomes preincubated with the ASL, suggesting that Domains 1 and 2 have roles in IRES binding to 40S subunits and that Domain 3 is located at the ribosome decoding site.     

Complete nucleotide sequence of carbazole/dioxin-degrading plasmid pCAR1 in Pseudomonas resinovorans strain CA10 indicates its mosaicity and the presence of large catabolic transposon Tn4676

The car and ant operons originally isolated from Pseudomonas resinovorans strain CA10 contain the genes encoding the carbazole/dioxin-degrading enzymes and anthranilate 1,2-dioxygenase, respectively, and are located on the plasmid pCAR1. The entire nucleotide sequence of pCAR1 was determined to elucidate the mechanism by which the car operon may have been assembled and distributed in nature. pCAR1 is a 199,035-bp circular plasmid, and carries 190 open reading frames. Although the incompatibility group of pCAR1 is unclear, its potential origin for replication, OriP, and Rep and Par proteins appeared to be closely related to those of plasmid pL6.5 isolated from Pseudomonas fluorescens. The potential tellurite-resistance klaABC genes identified in the neighboring region of repA gene were also related to those in IncP plasmid originally identified from pseudomonads. On the other hand, we found genes encoding proteins that showed low but significant homology (20-45% identity) with Trh and Tra proteins from Enterobacteriaceae, which are potentially involved in conjugative transfer of plasmids or genomic island, suggesting that pCAR1 is also a conjugative plasmid. In pCAR1, we found tnpAcCST genes that encoded the proteins showing >70% length-wise identities with those are encoded by the toluene/xylene-degrading transposon Tn4651 of TOL plasmid pWW0. Both car and ant degradative operons were found within a 72.8-kb Tn4676 sequence defined by flanking tnpAcC and tnpST genes and bordered by a 46-bp inverted repeat (IR). Within Tn4676 and its flanking region, we found the remnants of numerous mobile genetic elements, such as the duplicated transposase genes that are highly homologous to tnpR of Tn4653 and the multiple candidates of IRs for Tn4676 and Tn4653-like element. We also found distinct regions with high and low G+C contents within Tn4676, which contain an ant operon and car operon, respectively. These results suggested that multiple step assembly could have taken place before the current structure of Tn4676 had been captured.     

Can an arbitrary sequence evolve towards acquiring a biological function?

Abstract To explore the possibility that an arbitrary sequence can evolve towards acquiring functional role when fused with other pre-existing protein modules, we replaced the D2 domain of the fd-tet phage genome with the soluble random polypeptide RP3-42. The replacement yielded an fd-RP defective phage that is six-order magnitude lower infectivity than the wild-type fd-tet phage. The evolvability of RP3-42 was investigated through iterative mutation and selection. Each generation consists of a maximum of ten arbitrarily chosen clones, whereby the clone with highest infectivity was selected to be the parent clone of the generation that followed. The experimental evolution attested that, from an initial single random sequence, there will be selectable variation in a property of interest and that the property in question was able to improve over several generations. fd-7, the clone with highest infectivity at the end of the experimental evolution, showed a 240-fold increase in infectivity as compared to its origin, fd-RP. Analysis by phage ELISA using anti-M13 antibody and anti-T7 antibody revealed that about 37-fold increase in the infectivity of fd-7 was attributed to the changes in the molecular property of the single polypeptide that replaced the D2 domain of the g3p protein. This study therefore exemplifies the process of a random polypeptide generating a functional role in rejuvenating the infectivity of a defective bacteriophage when fused to some preexisting protein modules, indicating that an arbitrary sequence can evolve toward acquiring a functional role. Overall, this study could herald the conception of new perspective regarding primordial polypeptides in the field of molecular evolution.     

Roles of cathepsins in reperfusion-induced apoptosis in cultured astrocytes

Astrocytic apoptosis may play a role in the central nervous system injury. We previously showed that reperfusion of cultured astrocytes with normal medium after exposure to hydrogen peroxide (H(2)O(2))-containing medium causes apoptosis. This study examines the involvement of the lysosomal enzymes cathepsins B and D in the astrocytic apoptosis. Reperfusion after exposure to H(2)O(2) caused a marked increase in caspase-3 and cathepsin D activities and a marked decrease in cathepsin B activity. Pepstatin A, an inhibitor of cathepsin D, and acetyl-L-aspartyl-L-methionyl-L-glutaminyl-L-aspart-1-aldehyde (Ac-DMQD-CHO), a specific inhibitor of caspase-3, blocked the H(2)O(2)-induced decrease in cell viability and DNA ladder formation in cultured rat astrocytes. The (L-3-trans-(propylcarbamoyl)oxirane-2-carbonyl)-L-isoleucyl-L-proline methyl ester (CA074 Me), a specific inhibitor of cathepsin B, did not affect the H(2)O(2)-induced cell injury. On the other hand, CA074 Me decreased cell viability with DNA ladder formation when cultured in the presence of Ac-DMQD-CHO. This caspase-independent apoptosis was attenuated by the addition of the cathepsin D inhibitor pepstatin A. Caspase-3 like activity was markedly inhibited by Ac-DMQD-CHO and partially by pepstatin A. Pepstatin A and CA074 Me inhibited cathepsin B and cathepsin D activities, respectively, in the presence and absence of Ac-DMQD-CHO. These results suggest that cathepsins B and D are involved in astrocytic apoptosis: cathepsin D acts as a death-inducing factor upstream of caspase-3 and the caspase-independent apoptosis is regulated antagonistically by cathepsins B and D.     

Disruption of the human pathogenic yeast Candida albicans catalase gene decreases survival in mouse-model infection and elevates susceptibility to higher temperature and to detergents

Catalase-deficient strains of the human pathogenic yeast Candida albicans were constructed using the URA-blaster method. The disruptant was viable and grew normally in an ordinary culture condition, but became extremely sensitive to treatment with hydrogen peroxide. No catalase activity was observed in a catalase (CCT)-gene-disrupted strain, 1F5-4-1, suggesting that there were no other catalase or catalase-like enzymes in this yeast. The disruptant was shown to be sensitive to higher temperature and to low concentrations of SDS, NP-40, or Triton X-100. After a wild-type CCT gene was reintroduced into the disruptant, catalase activity was restored and the strain became moderately sensitive to treatment with hydrogen peroxide. However, neither the temperature sensitivity nor the susceptibility to SDS observed in the disruptant was restored in the CCT-reintroduced strain. A model infection experiment using wild-type and dCCT strains showed that the disruptants disappeared more rapidly than the wild-type strain in mouse liver, lung, and spleen. These results suggest that the catalase plays a significant role in survival in the host immune system and thus leads this organism to establish infection in the host.