Purification of Mesodermal-inducing Substances from Carp Swim Bladders I. Extraction and Isoelectric Focusing

Mesodermal-inducing substances were isolated from the swim bladders of carp ( Carassius auratus ). These substances, extracted with 8 M urea, can induce the development of early gastrula ectoderm cells of newts into mesodermal tissues, such as muscles, notochords, mesenchyme, renal tubules, and so on, to a high degree. Isoelectric focusing was used to isolate them as a first purifying step, and five component parts with different pH ranges were obtained. Each part was tested for mesodermal-inducing activity. On the basis of the extent and percentage of the induced tissues, two of them (parts C and D with pH ranges 6.0 to 7.0 and 7.0 to 9.0, respectively) were found to have high inducing activity, and the others had lower activity. The substances present in part C showed only low mesodermal-inducing activity, when diluted with gamma-globulin 100-fold. Apparently highly active mesodermal-inducing substances exist in the two parts C and D.     

Circadian Motile Activity of Erythrophores in the Red Abdominal Skin of Tetra Fishes and Its Possible Significance in Chromatic Adaptation

The red abdominal skin of the neon tetra Paracheirodon innesi and the cardinal tetra P. axelrodi was found to blanch at night or in the dark. Melatonin added to the bathing medium caused blanching of the red skin. Microscopic observations of the erythrophores indicated that the erythrosomes aggregated in response to norepinephrine, melanin-concentrating hormone (MCH), and melatonin. Of these compounds, melatonin was the most effective. By contrast, many erythrophores were refractory to MCH. Alpha-melanophore-stimulating hormone, isoproterenol, adenosine, and ATP each caused dispersal of the pigment to some extent. Isoproterenol dispersed the pigment only when an alpha-adrenergic blocker, tolazoline, was present. It appears that the change in color of the abdominal skin is primarily due to increased secretion during the night of the pineal hormone melatonin, while other hormonal and nervous factors may modify the distribution of the pigment in the erythrophores.     

A Cell Kinetic and Cytological Study on the Asymmetric Cell Division of Thymic Lymphoblasts of the Embryonic Rat

Cell division of thymus lymphoid cells from embrynonic and young rats was investigated cytologically on cell smears, focusing attention on asymmetric cell division. Some of thymic lymphoblasts displayed features implicating asymmetric cell division. At the telophase of such cells, two immature daughter cells looked dissimilar: one of them was smaller in size and possessed a more condensed nucleus, compared with the counterpart cell. Furthrmore, in most cases the cytoplasm of the smaller daughter cell was stained with Giemsa more deeply. It was suggested that the asymmetry of the nucleus emerges at anaphase and telophase probably due to some polarized situation of the cytoplasm. Asymmetrically-dividing cells were relatively frequently observed during the developmental period when large lymphoblasts actively transform into smaller lymphocytes :16% to 17% of whole dividing cells were under asymmetric cell division on days 16 and 17 of gestation, while less than 5% on day 19 or thereafter. In correlation with this observation, asymmetrically-dividing cells were more frequently observed among large lymphoblasts than among other smaller cell fractions. These results support the view that the asymmetric cell division may play some essential role in the transformation of large lymphoblasts into smaller lymphocytes.     

ROLE OF GLUCOCORTICOIDS IN TERMINAL DIFFERENTIATION AND TISSUE-SPECIFIC FUNCTION (A REVIEW)

Glucocorticoids (GCs) are required by many kinds of organs, tissues and cells for initiation or maintenance of their specific functions in vivo and in vitro. It is noticeable that most of these GC actions can be induced at much lower levels of dosage or concentration than the well-known actions of the steroids in gluconeogenesis, immune suppression and anti-inflammation. Such "differentiation-stimulating" actions of GC are regarded as main physiological roles of the steroid.     

S100 protein-immunoreactive trigeminal neurons innervating the rat molar tooth pulp

S100-immunoreactivity (ir) was examined in tooth pulp primary neurons of the rat. An immunofluorescence method demonstrated that the molar tooth pulp contained S100-immunoreactive (ir) nerve fibers. In the root pulp, pulp horn and roof of the pulp chamber, S100-ir smooth and varicose fibers ramified and formed subodontoblastic nerve plexuses. All the fibers became varicose at the base of the odontoblastic layer and extended to the odontoblastic layer. Some varicose endings could be traced into the dentin. The trigeminal neurons retrogradely labeled with fluorogold (FG) from the first and second maxillary molar tooth pulps exhibited S100- and parvalbumin-ir. Approximately 60% and 24% of the labeled cells were ir for S100 and parvalbumin, respectively. Virtually all parvalbumin-ir FG-labeled cells showed S100-ir, while 40% of S100-ir ones coexpressed parvalbumin-ir. An immunoelectron microscopic method revealed that all myelinated axons and half of the unmyelinated axons in the root pulp contained S100-ir. In the odontoblastic layer, predentin and dentin, S100-ir neurites lost the Schwann cell ensheathment and made close contact with cell bodies and processes of odontoblasts. The odontoblastic layer also contained parvalbumin-ir neurites. These neurites were devoid of the Schwann cell ensheathment and in close apposition to cell bodies and processes of odontoblasts. S100-ir pulpal axons seemed to be insensitive to repeated neonatal capsaicin treatment. This study suggests that S100-ir tooth pulp primary neurons are mostly myelinated and that S100-ir unmyelinated axons in the root pulp are preterminal segments of myelinated stem axons.     

ASC is an activating adaptor for NF-kappa B and caspase-8-dependent apoptosis

ASC is a pro-apoptotic protein containing a pyrin domain (PD) and a caspase-recruitment domain (CARD). A previous study suggests that ASC interacts with Ipaf, a member of the Apaf-1/Nod1 protein family. However, the functional relevance of the interaction has not been determined. Here, we report that co-expression of ASC with Ipaf or oligomerization of ASC induces both apoptosis and NF-kappa B activation. Apoptosis induced through ASC was inhibited by a mutant form of Caspase-8 but not by that of Caspase-1. The PD of ASC physically interacted with Caspase-8 as well as with pyrin, the familial Mediterranean fever gene product. Caspase-8 deficiency rescued mouse fibroblasts from apoptosis induced by ASC oligomerization. Pyrin disrupted the interaction between ASC and Caspase-8, and inhibited both apoptosis and NF-kappa B activation induced by ASC. These findings suggest that ASC is a mediator of NF-kappa B activation and Caspase-8-dependent apoptosis in an Ipaf signaling pathway.     

Exogenous cytosine deaminase gene expression in Bifidobacterium breve I-53-8w for tumor-targeting enzyme/prodrug therapy

Bifidobacteria are nonpathogenic, anaerobic domestic bacteria with health-promoting properties for the host. In our previous study, Bifidobacterium longum (B. longum) were found to be localized selectively and to proliferate within solid tumors after systemic application. Additionally, B. longum transformed by shuttle-plasmid including the cytosine deaminase (CD) gene expressed active CD, converted the prodrug 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU). We also demonstrated antitumor efficacy with a transformant of B. longum in rats. In this study, we found that Bifidobacterium breve (B. breve), the smallest species of human-derived bifidobacterium, expressed the exogenous transgene (CD), that CD enzymatic activity in the transformant of B. breve was much higher, and that the segregational stability of the plasmid was greater than that of B. longum. Thus, numerous transformants of B. breve were detected solely in the tumors after systemic administration. We consider the transformant of B. breve to be more beneficial in our enzyme/prodrug therapy.     

Crucial Role of Hyaluronan in Neointimal Formation after Vascular Injury

Background

Hyaluronan (HA) is a primary component of the extracellular matrix of cells, and it is involved in the pathogenesis of atherosclerosis. The purpose of this study was to investigate the role of HA in neointimal formation after vascular injury and determine its tissue-specific role in vascular smooth muscle cells (VSMCs) by using a cre-lox conditional transgenic (cTg) strategy.

Methods and Results

HA was found to be expressed in neointimal lesions in humans with atherosclerosis and after wire-mediated vascular injury in mice. Inhibition of HA synthesis using 4-methylumbelliferone markedly inhibited neointimal formation after injury. In vitro experiments revealed that low-molecular-weight HA (LMW-HA) induced VSMC activation, including migration, proliferation, and production of inflammatory cytokines, and reactive oxygen species (ROS). The migration and proliferation of VSMCs were mediated by the CD44/RhoA and CD44/ERK1/2 pathways, respectively. Because HA synthase 2 (HAS2) is predominantly expressed in injured arteries, we generated cTg mice that overexpress the murine HAS2 gene specifically in VSMCs (cHAS2/CreSM22α mice) and showed that HA overexpression markedly enhanced neointimal formation after cuff-mediated vascular injury. Further, HA-overexpressing VSMCs isolated from cHAS2/CreSM22α mice showed augmented migration, proliferation, and production of inflammatory cytokines and ROS.

Conclusion

VSMC-derived HA promotes neointimal formation after vascular injury, and HA may be a potential therapeutic target for cardiovascular disease.     

Translation of RNA from Eggs During Post-Diapause Development of Bombyx mori

Eggs of Bombyx mori are aroused from diapause by long-term chilling and develop when transferred to 25°C. During the first 20 hr of post-diapause development, the polysome content and the presumed rate of protein synthesis increase about 3-fold, while the ribosome content and the total RNA content increase only 1.1-fold. In this study, total RNAs were extracted from chilled eggs (termed 0 hr of development), and post-diapause eggs at 10 and 20 hr of development. The RNAs were purified further by high pressure liquid chromatography to remove RNA-like oligonucleotides. On translation in a protein-synthesizing system derived from wheat germ with a subsaturating amount of RNA, no difference was found in the relative amounts of translatable mRNA activity at 10 and 20 hr of development from that at 0 hr. Moreover, the translation products of the different RNA preparations in a rabbit reticulocyte lysate system appeared very similar when separated by gel electrophoresis and located by fluorography. These facts suggest that protein synthesis in early post-diapause development is controlled at a translational level.