Effects of RNA interference of Atg4B on the limited proteolysis of LC3 in PC12 cells and expression of Atg4B in various rat tissues

Atg4B, a mammalian homologue of yeast Atg4, has been shown to play an important role in the processing of LC3, a mammalian homologue of yeast Atg8, but the tissue distribution of Atg4B remains unknown. To better understand the role of Atg4B in rat tissue cells, we prepared antibodies against Atg4B, and PC12 cells in which the expression of Atg4B was knocked down by RNA interference. In the RNA interference-treated PC12 cells, for which the expression of Atg4B was 10% of wild-type PC12 cells, the expression of cytosolic LC3-I was similar to that in wild-type cells. Knockdown cell lysates, however, suppressed the cleavage of recombinant proLC3 to LC3-I. Moreover, the expression of Atg4B protein and mRNA was ubiquitous in rat tissues; however, the expression levels were not identical, but were dependent on the tissue, with the expression high in brain and testicular tissue, and low in muscular and heart tissue. In brain tissue, the expression of Atg4B protein and mRNA was higher in neurons, especially in the cerebellum and olfactory bulb, as evidenced by immunohistochemistry and in situ hybridization. These lines of evidence suggest that Atg4B plays a major role in the processing of LC3 and is widely distributed in rat tissues. In particular, in brain tissues, autophagy may be deeply associated with the metabolism of neurons, especially in the cerebellum.     

Introgression of Drosophila simulans Nuclear Pore Protein 160 in Drosophila melanogaster Alone Does Not Cause Inviability but Does Cause Female Sterility

We have been analyzing genes for reproductive isolation by replacing Drosophila melanogaster genes with homologs from Drosophila simulans by interspecific backcrossing. Among the introgressions established, we found that a segment of the left arm of chromosome 2, Int(2L)S, carried recessive genes for hybrid sterility and inviability. That nuclear pore protein 160 (Nup160) in the introgression region is involved in hybrid inviability, as suggested by others, was confirmed by the present analysis. Male hybrids carrying an X chromosome of D. melanogaster were not rescued by the Lethal hybrid rescue (Lhr) mutation when the D. simulans Nup160 allele was made homozygous or hemizygous. Furthermore, we uniquely found that Nup160 is also responsible for hybrid sterility. Females were sterile when D. simulans Nup160 was made homozygous or hemizygous in the D. melanogaster genetic background. Genetic analyses indicated that the D. simulans Nup160 introgression into D. melanogaster was sufficient to cause female sterility but that other autosomal genes of D. simulans were also necessary to cause lethality. The involvement of Nup160 in hybrid inviability and female sterility was confirmed by transgene experiment.INVESTIGATING the genetic bases of reproductive isolation is important for understanding speciation (Sawamura and Tomaru 2002; Coyne and Orr 2004; Wu and Ting 2004; Noor and Feder 2006; Presgraves 2010). In fact, continued interest in this issue has led to the isolation of several genes that are responsible for hybrid sterility and inviability in Drosophila (Ting et al. 1998; Barbash et al. 2003; Presgraves et al. 2003; Brideau et al. 2006; Masly et al. 2006; Phadnis and Orr 2009; Prigent et al. 2009; Tang and Presgraves 2009). Drosophila melanogaster and Drosophila simulans are the best pair for such genetic analyses (Sturtevant 1920). Hybrid male lethality in the cross between D. melanogaster females and D. simulans males is caused by incompatibility involving chromatin-binding proteins (Barbash et al. 2003; Brideau et al. 2006), and hybrid female lethality in the reciprocal cross is caused by incompatibility between a maternally supplied factor and a repetitive satellite DNA (Sawamura et al. 1993a; Sawamura and Yamamoto 1997; Ferree and Barbash 2009). Furthermore, individuals with the genotype equivalent to the backcrossed generation exhibit different incompatibilities (Pontecorvo 1943; Presgraves 2003), two components of which have been identified (Presgraves et al. 2003; Tang and Presgraves 2009). Because of the discovery of rescuing mutations that prevent hybrid inviability and sterility (Watanabe 1979; Hutter and Ashburner 1987; Sawamura et al. 1993a,b; Davis et al. 1996; Barbash and Ashburner 2003), chromosome segments from D. simulans can be introgressed into the D. melanogaster genome (Sawamura et al. 2000; Masly et al. 2006). For example, introgression of the D. simulans chromosome 4 or Y into D. melanogaster results in male sterility (Muller and Pontecorvo 1940; Orr 1992), and the recessive sterility by the chromosome 4 introgression is attributed to an interspecific gene transposition between chromosomes (Masly et al. 2006).The other successful introgressions of this type are the tip and the middle regions of the left arm of chromosome 2, Int(2L)D and Int(2L)S, respectively (Sawamura et al. 2000). Both female and male Int(2L)S homozygotes are sterile (Figure 1A), and the recessive sterility genes have been mapped with recombination and complementation assays against deficiencies. The male sterility genes are polygenic and interact epistatically with each other (Sawamura and Yamamoto 2004; Sawamura et al. 2004b), but the female sterility gene has been mapped to a 170-kb region containing only 20 open reading frames (ORFs) (Sawamura et al. 2004a). Interestingly, Int(2L)S also carries a recessive lethal gene whose effect is detected only in a specific genotype (Figure 1B). Lethality in hybrid males from the cross between D. melanogaster females and D. simulans males is rescued by the Lethal hybrid rescue (Lhr) mutation in D. simulans (Watanabe 1979), but the hybrid males cannot be rescued if they carry the introgression, presumably because of incompatibility between an X-linked gene(s) of D. melanogaster and a homozygous D. simulans gene in the Int(2L)S region (Sawamura 2000). Because the female sterility gene and the lethal gene were not separated by recombination, Sawamura et al. (2004a) suggested that female sterility and lethality may be a consequence of the pleiotropic effects of a single gene.Open in a separate windowFigure 1.—Viability and fertility of flies with various genotypes. (A) Females and males that are heterozygous or homozygous for the D. simulans introgression Int(2L)S (Int) in the D. melanogaster genetic background. (B) Four genotypic classes from the cross between introgression heterozygote [Int(2L)S/CyO] females and D. simulans Lethal hybrid rescue (Lhr) males. (C) Four genotypic classes from the cross between D. melanogaster females with a deficiency (Df) [Df(2L)/CyO] and D. simulans Lhr males. Open chromosome regions are from D. melanogaster, and shaded ones are from D. simulans.Because the hybrid lethal gene on Int(2L)S is recessive, the gene can be mapped by deficiencies instead of using introgression (Figure 1C) (Sawamura 2000; Sawamura et al. 2004a). In fact, hybrid males carrying a deficiency encompassing this region (hemizygous for the D. simulans genes) and the D. melanogaster X chromosome are lethal even if they carry the hybrid rescue mutation (see also Presgraves 2003). Tang and Presgraves (2009) subsequently narrowed down this region with multiple deficiencies and identified the hybrid lethal gene with a complementation test and transformation. We confirmed their conclusion and report our data here. In the transformation experiment, we used the natural promoter of the gene, instead of overexpressing the gene (Tang and Presgraves 2009), and we directly indicated, for the first time, that the hybrid lethal gene is also responsible for the female sterility of introgression homozygotes. The D. simulans allele of the gene seems to be nonfunctional on the genetic background of D. melanogaster. Moreover, our results indicated that this gene and chromosome X of D. melanogaster are not sufficient to explain the inviability and that another autosomal gene(s) in D. simulans is required.     

Characterization of apparently balanced chromosomal rearrangements from the developmental genome anatomy project

Apparently balanced chromosomal rearrangements in individuals with major congenital anomalies represent natural experiments of gene disruption and dysregulation. These individuals can be studied to identify novel genes critical in human development and to annotate further the function of known genes. Identification and characterization of these genes is the goal of the Developmental Genome Anatomy Project (DGAP). DGAP is a multidisciplinary effort that leverages the recent advances resulting from the Human Genome Project to increase our understanding of birth defects and the process of human development. Clinically significant phenotypes of individuals enrolled in DGAP are varied and, in most cases, involve multiple organ systems. Study of these individuals' chromosomal rearrangements has resulted in the mapping of 77 breakpoints from 40 chromosomal rearrangements by FISH with BACs and fosmids, array CGH, Southern-blot hybridization, MLPA, RT-PCR, and suppression PCR. Eighteen chromosomal breakpoints have been cloned and sequenced. Unsuspected genomic imbalances and cryptic rearrangements were detected, but less frequently than has been reported previously. Chromosomal rearrangements, both balanced and unbalanced, in individuals with multiple congenital anomalies continue to be a valuable resource for gene discovery and annotation.     

Algicidal activity of polyunsaturated fatty acids derived from <Emphasis Type="Italic">Ulva fasciata</Emphasis> and <Emphasis Type="Italic">U. pertusa</Emphasis> (Ulvaceae,Chlorophyta) on phytoplankton

Isolation of algicidal compounds from Ulva fasciata revealed that the algicidal substances were the polyunsaturated fatty acids (PUFAs) as hexadeca-4,7,10,13-tetraenoic acid (HDTA) C16:4 n-3, octadeca-6,9,12,15-tetraenoic acid (ODTA) C18:4 n-3, α-linolenic acid (ALA) C18:3 n-3 and linoleic acid (LA) C18:2 n-6. The fatty acid composition of four species of Ulvaceae (U. fasciata, U. pertusa, U. arasakii and U. conglobota) was analyzed by capillary gas chromatography to investigate the relationship with the algicidal activity. The results indicate that highly algicidal species, U. fasciata and U. pertusa, showed higher contents of C16:4 n-3, C18:3 n-3, and C18:4 n-3. Concentrations of these PUFAs released from the seaweed in the culture medium were also analyzed. These PUFAs were found to be significantly active against Chattonella antiqua, C. marina, Fibrocapsa japonica, Heterosigma akashiwo, Karenia mikimotoi, moderately effective against Heterocapsa circularisquama, Prorocentrum minimum, P. sigmoides, Scrippsiella trochoidea, whereas low effective against Alexandrium catenella and Cochlodinium polykrikoides. It is suggested that the PUFAs are useful mitigation agents to remove several harmful effects without causing detrimental effects on surrounding marine living organisms.     

Design and synthesis of a bis(cycloisodityrosine) analogue of RA-VII, an antitumor bicyclic hexapeptide

An analogue of an antitumor bicyclic hexapeptide RA-VII was prepared, in which the Ala-2 and Tyr-3 residues of RA-VII were replaced by a cycloisodityrosine unit. In the crystalline state, the peptide backbone structures and the side-chain conformations at Tyr-3, Tyr-5, and Tyr-6 of this analogue and of RA-II were very similar. This analogue, however, showed much weaker cytotoxicity against P-388 leukemia cells than parent RA-VII.     

Identification of galectin-3-binding protein as a factor secreted by tumor cells that stimulates interleukin-6 expression in the bone marrow stroma

We have previously demonstrated that neuroblastoma cells increase the expression of interleukin-6 by bone marrow stromal cells and that stimulation does not require cell-cell contact. In this study we report the purification and identification of a protein secreted by neuroblastoma cells that stimulates interleukin-6 production by stromal cells. Using a series of chromatographic purification steps including heparin-affinity, ion exchange, and molecular sieve chromatography followed by trypsin digestion and liquid chromatography tandem mass spectrometry, we identified in serum-free conditioned medium of neuroblastoma cells several secreted peptides including galectin-3-binding protein, also known as 90-kDa Mac-2-binding protein. We demonstrated the presence of the galectin-3-binding protein in the conditioned medium of several neuroblastoma cell lines and in chromatographic fractions with interleukin-6 stimulatory activity. Consistently, bone marrow stromal cells express galectin-3, the receptor for galectin-3-binding protein. Supporting a role for galectin-3-binding protein in stimulating interleukin-6 expression in bone marrow stromal cells, we observed that recombinant galectin-3-binding protein stimulated interleukin-6 expression in these cells and that interleukin-6 stimulation by neuroblastoma-conditioned medium was inhibited in the presence of lactose or a neutralizing anti-galectin-3 antibody. Down-regulation of galectin-3-binding protein expression in neuroblastoma cells also decreased the interleukin-6 stimulatory activity of the conditioned medium on bone marrow stromal cells. We also provide evidence that stimulation of interleukin-6 by galectin-3-binding protein involves activation of the Erk1/2 pathway. The data, thus, identifies galectin-3-binding protein as a factor secreted by neuroblastoma cells that stimulates the expression of interleukin-6 in bone marrow stromal cells and provides a novel function for this protein in cancer progression.     

Lysophosphatidic acid and autotaxin stimulate cell motility of neoplastic and non-neoplastic cells through LPA1

Autotaxin (ATX) is a tumor cell motility-stimulating factor originally isolated from melanoma cell supernatant that has been implicated in regulation of invasive and metastatic properties of cancer cells. Recently, we showed that ATX is identical to lysophospholipase D, which converts lysophosphatidylcholine to a potent bioactive phospholipid mediator, lysophosphatidic acid (LPA), raising the possibility that autocrine or paracrine production of LPA by ATX contributes to tumor cell motility. Here we demonstrate that LPA and ATX mediate cell motility-stimulating activity through the LPA receptor, LPA(1). In fibroblasts isolated from lpa(1)(-/-) mice, but not from wild-type or lpa(2)(-/-), cell motility stimulated with LPA and ATX was completely absent. In the lpa(1)(-/-) cells, LPA-stimulated lamellipodia formation was markedly diminished with a concomitant decrease in Rac1 activation. LPA stimulated the motility of multiple human cancer cell lines expressing LPA(1), and the motility was attenuated by an LPA(1)-selective antagonist, Ki16425. The present study suggests that ATX and LPA(1) represent potential targets for cancer therapy.