Molecular diversity of skin mucus lectins in fish

Among lectins in the skin mucus of fish, primary structures of four different types of lectin have been determined. Congerin from the conger eel Conger myriaster and AJL-1 from the Japanese eel Anguilla japonica were identified as galectin, characterized by its specific binding to β-galactoside. Eel has additionally a unique lectin, AJL-2, which has a highly conserved sequence of C-type lectins but displays Ca²⁺-independent activity. This is rational because the lectin exerts its function on the cutaneous surface, which is exposed to a Ca²⁺ scarce environment when the eel is in fresh water. The third type lectin is pufflectin, a mannose specific lectin in the skin mucus of pufferfish Takifugu rubripes. This lectin showed no sequence similarity with any known animal lectins but, surprisingly, shares sequence homology with mannose-binding lectins of monocotyledonous plants. The fourth lectin was found in the ponyfish Leiognathus nuchalis and exhibits homology with rhamnose-binding lectins known in eggs of some fish species. These lectins, except ponyfish lectin, showed agglutination of certain bacteria. In addition, pufflectin was found to bind to a parasitic trematode, Heterobothrium okamotoi. Taken together, these results demonstrate that skin mucus lectins in fish have wide molecular diversity.     

Synthesis of antisense oligonucleotides carrying modified 2-5A molecules at their 5'-termini and their properties

The synthesis of 8-methyladenosine-substituted 2-5A tetramers with hydroxyalkyl groups at the 5'-phosphates and the corresponding 2-5A-antisense chimeras is described. These oligonucleotides were synthesized by the phosphoramidite method with a DNA/RNA synthesizer. These 2-5A tetramers with hydroxyethyl and hydroxybutyl groups at their 5'-phosphates were more resistant to hydrolysis by alkaline phosphatase than those without the hydroxyalkyl groups. Incorporation of the hydroxyethyl group into the 2-5A tetramer and 2-5A-antisense chimera slightly reduced the abilities of their analogues to activate recombinant human RNase L, but the abilities of the 2-5A tetramer and the 2-5A-antisense chimera both with the hydroxyethyl group and 8-methyladenosine returned to 80 and 50% relative to those of the oligonucleotides without the hydroxyethyl group and 8-methyladenosine, respectively. Furthermore, the enzyme activated by 8-methyladenosine-substituted 2-5A-antisense chimera with the hydroxyethyl group cleaved the complementary RNA as efficiently as that activated by 2-5A-antisense chimera without the hydroxyethyl group and 8-methyladenosine. Thus, the 2-5A-antisense chimera carrying the hydroxyethyl group and 8-methyladenosine will be a candidate for a novel antisense molecule.     

Biosynthesis and action of neurosteroids in the cerebellar Purkinje neuron

The brain is considered to be a target site of peripheral steroid hormones. In contrast to this classical concept, new findings over the past decade have established that the brain itself also synthesizes steroids de novo from cholesterol through mechanisms at least partly independent of peripheral steroidogenic glands. Such steroids synthesized de novo in the brain, as well as other areas of the nervous system, are called neurosteroids. To understand neurosteroid actions in the brain, we need data on the specific synthesis in particular sites of the brain at particular times. Therefore, our studies for this exciting area of brain research have focused on the biosynthesis and action of neurosteroids in the identified neurosteroidogenic cells underlying important brain functions. We have demonstrated that the Purkinje cell, a typical cerebellar neuron, is a major site for neurosteroid formation in the brain. This is the first observation of neuronal neurosteroidogenesis in the brain. Subsequently, genomic and nongenomic actions of neurosteroids have become clear by a series of our studies using an excellent Purkinje cellular model. On the basis of these findings, we summarize the advances made in our understanding of biosynthesis and action of neurosteroids in the cerebellar Purkinje cell.     

Light-sensitive voltage responses in the neurons of the cerebral ganglion of Ciona savignyi (Chordata: Ascidiacea)

Light-responsive behaviors such as siphon contraction (1), phototropism (2), and gamete release (3, 4) have been described in several ascidian species. The pigmented spots around the siphon openers (5), the epithelial cells of the sperm duct (6, 7), and the cerebral ganglion (8) have been suggested to be the photoreceptor candidates underlying these behaviors. However, these arguments have not yet been settled because no direct electrophysiological recordings of light-induced receptor potentials have been reported. In this study, we focused on the cerebral ganglion and performed intracellular recordings from the neurons in the ventral side of the cerebral ganglion in an isolated in vitro preparation of the neural complex in Ciona savignyi. We found that 24% (n = 115) of the recorded neurons showed various types of voltage responses to light stimuli. Almost all (27/28) of the recorded voltage responses were "on" responses that included hyperpolarizing and depolarizing responses and could be categorized into five types, except for a complex response recorded in one cell; the remaining one (1/28) was a depolarizing "off" response. This is the first report of electrophysiological recordings of light-sensitive voltage responses from ascidian cerebral ganglion neurons.     

Skin-specific caspase-1-transgenic mice show cutaneous apoptosis and pre-endotoxin shock condition with a high serum level of IL-18

To study the pathophysiological roles of overexpressed caspase-1 (CASP1), originally designated as IL-1 beta-converting enzyme, we generated transgenic mice in which human CASP1 is overexpressed in their keratinocytes. The transgenic mice spontaneously developed recalcitrant dermatitis and skin ulcers, characterized by the presence of massive keratinocyte apoptosis. The skin of the mice contained the active form of human CASP1 and expressed mRNA for caspase-activated DNase, an effector endonuclease responsible for DNA fragmentation. Their skin and sera showed elevated levels of mature IL-18 and IL-1 beta, but not of IFN-gamma. The plasma from these animals induced IFN-gamma production by IL-18-responsive NK cells. Administration of heat-killed Propionibacterium acnes, a potent in vivo type 1 cell inducer, caused IFN-gamma-mediated lethal liver injury in the transgenic mice, which was completely inhibited by treatment with neutralizing anti-IL-18 Ab. These results indicated that in vivo overexpression of CASP1 caused spontaneous apoptotic tissue injury and rendered mice highly susceptible to exogenous type 1 cell-inducing condition in collaboration with endogenously accumulated proinflammatory cytokines.     

Growth retardation and microcephaly induced in mice by placental infection with murine cytomegalovirus

BACKGROUND: The placenta is regarded as a site of congenital cytomegalovirus (CMV) infection. The placental infection of fetuses with murine CMV (MCMV) was investigated in a mouse model. METHODS: The placentas and fetuses were examined using the polymerase chain reaction (PCR) and Southern blotting for viral DNA and immunostaining for viral antigen. Since the transplacental infection rarely occurs, the placentas were directly injected with MCMV at day 12.5 of gestation; the embryos were then allowed to develop until day 18.5 of gestation. RESULTS: Formation of infected foci at day 18. 5 of gestation was found in more than 60% of the injected placentas. Infection of about 50% of the embryos occurred from the infected placentas. The frequency of infection in the brain was 27%, which was the same as that in the liver and higher than that in the lungs. In the brains, infected cells were often observed in the ventricular zone of the cerebrum and sometimes in the cortical plate and the hippocampus. Developmental retardation with microcephaly was observed in about 25% of offspring exposed to infection in utero. CONCLUSIONS: These results suggest that formation of infected foci in the placenta is important for embryonic congenital infection, and that the cerebral ventricular zone is one of the most susceptible sites for CMV infection in the embryonic stage.     

Amitrole-induced cell transformation and gene mutations in Syrian hamster embryo cells in culture

Amitrole, a widely used herbicide, is an animal carcinogen and an inducer of cell transformation. However, it is inactive as a mutagen in bacterial test systems. Thus, it has been suggested that amitrole is a non-mutagenic carcinogen. Over the dose range that induces morphological transformation of Syrian hamster embryo cells in culture, amitrole induced gene mutations at the Na+/K+ ATPase and hypoxanthine phosphoribosyl transferase loci measured concomitantly in the same cells. These results indicate that amitrole may act via a mutational mechanism.     

Melting Curve Analysis after T Allele Enrichment (MelcaTle) as a Highly Sensitive and Reliable Method for Detecting the JAK2V617F Mutation

Detection of the JAK2V617F mutation is essential for diagnosing patients with classical myeloproliferative neoplasms (MPNs). However, detection of the low-frequency JAK2V617F mutation is a challenging task due to the necessity of discriminating between true-positive and false-positive results. Here, we have developed a highly sensitive and accurate assay for the detection of JAK2V617F and named it melting curve analysis after T allele enrichment (MelcaTle). MelcaTle comprises three steps: 1) two cycles of JAK2V617F allele enrichment by PCR amplification followed by BsaXI digestion, 2) selective amplification of the JAK2V617F allele in the presence of a bridged nucleic acid (BNA) probe, and 3) a melting curve assay using a BODIPY-FL-labeled oligonucleotide. Using this assay, we successfully detected nearly a single copy of the JAK2V617F allele, without false-positive signals, using 10 ng of genomic DNA standard. Furthermore, MelcaTle showed no positive signals in 90 assays screening healthy individuals for JAK2V617F. When applying MelcaTle to 27 patients who were initially classified as JAK2V617F-positive on the basis of allele-specific PCR analysis and were thus suspected as having MPNs, we found that two of the patients were actually JAK2V617F-negative. A more careful clinical data analysis revealed that these two patients had developed transient erythrocytosis of unknown etiology but not polycythemia vera, a subtype of MPNs. These findings indicate that the newly developed MelcaTle assay should markedly improve the diagnosis of JAK2V617F-positive MPNs.