A novel human hepatoma cell line, FLC-4, exhibits highly enhanced liver differentiation functions through the three-dimensional cell shape

We characterized three-dimensional human hepatoma cell lines, functional liver cell (FLC) cell lines, to establish a highly differentiated hepatoma cell line. We investigated the effect of extracellular matrix and cell morphology on liver-specific gene expression in FLC cells. The hepatocyte nuclear factor-4α (HNF-4α) and other liver-specific gene expressions were enhanced in spherical FLC-4 cells on EHS-gel, but other human hepatoma cells such as HepG2 did not show the enhancement. Importantly, the liver-specific gene expression levels in spherical FLC-4 cells cultured on EHS-gel were comparable to those of human liver and were much higher than those of other human hepatoma cell lines. The major matrix components and growth factors in EHS-gel did not affect cell shape and liver functions. To exclude any effect of the extracellular matrix, we made spherical FLC-4 cells by actin filament disruption. The actin-disrupted spherical cells also showed an enhanced liver-specific gene expression. We concluded that three-dimensional cell shape per se is one of the most important determinants of liver differentiation functions in FLC-4 cells. Cell morphology-dependent induction of liver-specific gene expression was mediated through microtubule organization. In conclusion, differentiation of FLC-4 human hepatoma cell line can be enhanced to a human liver-like level through the three-dimensional cell shape in a microtubule-dependent manner.     

Experimental challenges of wild Manila clams with Perkinsus species isolated from naturally infected wild Manila clams

Manila clams, Ruditapes philippinarum, are widely harvested in the coastal waters in Japan. However, there have been significant decreases in the populations of Manila clams since the 1980s. It is thought that infection with the protozoan Perkinsus species has contributed to these decreases. A previous study demonstrated that high infection levels of a pure strain of Perkinsus olseni (ATCC PRA-181) were lethal to hatchery-raised small Manila clams, however, the pathogenicity of wild strain Perkinsus species to wild Manila clam is unclear. To address this, we challenged large (30-40mm in shell length) and small (3-15mm in shell length) wild Manila clams with Perkinsus species isolated from naturally infected wild Manila clams. We report high mortalities among the small clams, but not among the large ones. This is the first report to confirm the pathogenicity of wild isolate of Perkinsus species to wild Manila clams.     

Sphingosine-1-phosphate lyase expression in embryonic and adult murine tissues

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid involved in immunity, inflammation, angiogenesis, and cancer. S1P lyase (SPL) is the essential enzyme responsible for S1P degradation. SPL augments apoptosis and is down-regulated in cancer. SPL generates a S1P chemical gradient that promotes lymphocyte trafficking and as such is being targeted to treat autoimmune diseases. Despite growing interest in SPL as a disease marker, antioncogene, and pharmacological target, no comprehensive characterization of SPL expression in mammalian tissues has been reported. We investigated SPL expression in developing and adult mouse tissues by generating and characterizing a β-galactosidase-SPL reporter mouse combined with immunohistochemistry, immunoblotting, and enzyme assays. SPL was expressed in thymic and splenic stromal cells, splenocytes, Peyer's Patches, colonic lymphoid aggregates, circulating T and B lymphocytes, granulocytes, and monocytes, with lowest expression in thymocytes. SPL was highly expressed within the CNS, including arachnoid lining cells, spinal cord, choroid plexus, trigeminal nerve ganglion, and specific neurons of the olfactory bulb, cerebral cortex, midbrain, hindbrain, and cerebellum. Expression was detected in brown adipose tissue, female gonads, adrenal cortex, bladder epithelium, Harderian and preputial glands, and hair follicles. This unique expression pattern suggests SPL has many undiscovered physiological functions apart from its role in immunity.     

Complement C1q activates canonical Wnt signaling and promotes aging-related phenotypes

Wnt signaling plays critical roles in development of various organs and pathogenesis of many diseases, and augmented Wnt signaling has recently been implicated in mammalian aging and aging-related phenotypes. We here report that complement C1q activates canonical Wnt signaling and promotes aging-associated decline in tissue regeneration. Serum C1q concentration is increased with aging, and Wnt signaling activity is augmented during aging in the serum and in multiple tissues of wild-type mice, but not in those of C1qa-deficient mice. C1q activates canonical Wnt signaling by binding to Frizzled receptors and subsequently inducing C1s-dependent cleavage of the ectodomain of Wnt coreceptor low-density lipoprotein receptor-related protein 6. Skeletal muscle regeneration in young mice is inhibited by exogenous C1q treatment, whereas aging-associated impairment of muscle regeneration is restored by C1s inhibition or C1qa gene disruption. Our findings therefore suggest the unexpected role of complement C1q in Wnt signal transduction and modulation of mammalian aging.     

Metal ion dependency of serine racemase from Dictyostelium discoideum

D-Serine is known to act as an endogenous co-agonist of the N-methyl-D-aspartate receptor in the mammalian brain and is endogenously synthesized from L-serine by a pyridoxal 5'-phosphate-dependent enzyme, serine racemase. Though the soil-living mycetozoa Dictyostelium discoideum possesses no genes homologous to that of NMDA receptor, it contains genes encoding putative proteins relating to the D-serine metabolism, such as serine racemase, D-amino acid oxidase, and D-serine dehydratase. D. discoideum is an attractive target for the elucidation of the unknown functions of D-serine such as a role in cell development. As part of the elucidation of the role of D-serine in D. discoideum, we cloned, overexpressed, and examined the properties of the putative serine racemase exhibiting 46% amino acid sequence similarity with the human enzyme. The enzyme is unique in its stimulation by monovalent cations such as Na(+) in addition to Mg(2+) and Ca(2+), which are well-known activators for the mammalian serine racemase. Mg(2+) or Na(+) binding caused two- to ninefold enhancement of the rates of both racemization and dehydration. The half-maximal activation concentrations of Mg(2+) and Na(+) were determined to be 1.2?μM and 2.2?mM, respectively. In the L-serine dehydrase reaction, Mg(2+) and Na(+) enhanced the k (cat) value without changing the K (m) value. Alanine mutation of the residues E207 and D213, which correspond to the Mg(2+)-binding site of Schizosaccharomyces pombe serine racemase, abolished the Mg(2+)- and Na(+)-dependent stimulation. These results suggest that Mg(2+) and Na(+) share the common metal ion-binding site.     

Reduced BMP Signaling Results in Hindlimb Fusion with Lethal Pelvic/Urogenital Organ Aplasia: A New Mouse Model of Sirenomelia

Sirenomelia, also known as mermaid syndrome, is a developmental malformation of the caudal body characterized by leg fusion and associated anomalies of pelvic/urogenital organs including bladder, kidney, rectum and external genitalia. Most affected infants are stillborn, and the few born alive rarely survive beyond the neonatal period. Despite the many clinical studies of sirenomelia in humans, little is known about the pathogenic developmental mechanisms that cause the complex array of phenotypes observed. Here, we provide new evidences that reduced BMP (Bone Morphogenetic Protein) signaling disrupts caudal body formation in mice and phenocopies sirenomelia. Bmp4 is strongly expressed in the developing caudal body structures including the peri-cloacal region and hindlimb field. In order to address the function of Bmp4 in caudal body formation, we utilized a conditional Bmp4 mouse allele (Bmp4^flox/flox) and the Isl1 (Islet1)-Cre mouse line. Isl1-Cre is expressed in the peri-cloacal region and the developing hindimb field. Isl1Cre;Bmp4^flox/flox conditional mutant mice displayed sirenomelia phenotypes including hindlimb fusion and pelvic/urogenital organ dysgenesis. Genetic lineage analyses indicate that Isl1-expressing cells contribute to both the aPCM (anterior Peri-Cloacal Mesenchyme) and the hindlimb bud. We show Bmp4 is essential for the aPCM formation independently with Shh signaling. Furthermore, we show Bmp4 is a major BMP ligand for caudal body formation as shown by compound genetic analyses of Bmp4 and Bmp7. Taken together, this study reveals coordinated development of caudal body structures including pelvic/urogenital organs and hindlimb orchestrated by BMP signaling in Isl1-expressing cells. Our study offers new insights into the pathogenesis of sirenomelia.     

Structural and functional characterization of <Emphasis Type="Italic">IbMYB1</Emphasis> genes in recent Japanese purple-fleshed sweetpotato cultivars

To elucidate further the genetic mechanism underlying anthocyanin accumulation in the storage roots of recent Japanese purple-fleshed sweetpotato cultivars, we compared the structure of the IbMYB1 gene in cultivar Ayamurasaki and its spontaneous mutant, AYM96, whose storage roots do not accumulate anthocyanin. Amplification of the IbMYB1 genomic fragment covering the coding sequences suggested that the genome of Ayamurasaki contained three types of IbMYB1 sequences, named IbMYB1-1, IbMYB1-2a and IbMYB1-2b, whereas AYM96 had only IbMYB1-1. Although these three IbMYB1 sequences had identical coding sequences, IbMYB1-1 had a 7-bp insertion in the first intron. IbMYB1-2a and IbMYB1-2b were characterized by a single nucleotide polymorphism in the second intron. Further cloning and sequencing of the flanking regions of these IbMYB1 sequences showed that the promoter and 3′ flanking regions of IbMYB1-2a and IbMYB1-2b were different from those of IbMYB1-1. Genetic analysis using an F₁ population derived from a cross between the purple-fleshed cultivar Murasakimasari and AYM96 suggested that IbMYB1-2 sequences are responsible for anthocyanin accumulation in the storage roots. The structural features of these three IbMYB1 sequences and identification of the IbMYB1-2null sequence, which contained sequences very similar to those of the flanking regions of IbMYB1-2a and IbMYB1-2b, but which lacked the sequence around the coding region, suggested that IbMYB1 genes in recent Japanese purple-fleshed cultivars had been established through multiple gene-duplication events.     

Assessment of hybrid vigor between flightless lines to restore survival and reproductive characteristics in the ladybird beetle <Emphasis Type="Italic">Harmonia axyridis</Emphasis>

The effectiveness of hybrid vigor, which can counteract deleterious effects of inbreeding in flightless Harmonia axyridis Pallas (Coleoptera: Coccinellidae), was investigated. First, we performed the reciprocal cross between two isofemale lines and compared survival and reproductive characteristics between isofemale and hybrid lines under laboratory conditions. The survival of one of the hybrid lines was significantly higher than that of the two isofemale lines. Early fecundity tended to be higher in the two hybrid lines than in the two isofemale lines. Second, we compared the effectiveness of control of Aphis gossypii Glover (Homoptera: Aphididae) between isofemale and hybrid lines by release experiments in greenhouses. The number of A. gossypii was suppressed in treatments in which two hybrid lines were released compared with those in which two isofemale lines were released. These results suggest that hybrid vigor is effective as a method for assuring the quality of flightless H. axyridis.