Ablation of Csk in neural crest lineages causes corneal anomaly by deregulating collagen fibril organization and cell motility

Src family kinases (SFKs) have been implicated in the regulation of cell motility. To verify their in vivo roles during development, we generated mutant mice in which Csk, a negative regulator of SFKs, was inactivated in neural crest lineages using the Protein zero promoter in a Cre-loxP system. Inactivation of Csk caused deformities in various tissues of neural crest origins, including facial dysplasia and corneal opacity. In the cornea, the stromal collagen fibril was disorganized and there was an overproduction of collagen 1a1 and several metalloproteases. The corneal endothelium failed to overlie the central region of the eye and the peripheral endothelium displayed a disorganized cytoskeleton. Corneal mesenchymal cells cultured from mutant mice showed attenuated cell motility. In these cells, p130 Crk-associated substrate (Cas) was hyperphosphorylated and markedly downregulated. The expression of a dominant negative Cas (CasΔSD) could suppress the cell motility defects. Fluorescence resonance energy transfer analysis revealed that activation of Rac1 and Cdc42 was depolarized in Csk-inactivated cells, which was restored by the expression of either Csk or CasΔSD. These results demonstrate that the SFKs/Csk circuit plays crucial roles in corneal development by controlling stromal organization and by ensuring cell motility via the Cas-Rac/Cdc42 pathways.     

Biosynthetic processing of human interleukin-2 receptor (Tac antigen)

Biosynthetic processing of the T-cell surface receptor for interleukin-2 was investigated in a cultured human T-cell line MT-1 by means of metabolic and cell surface radiolabeling followed by immunoprecipitation with a monoclonal anti-receptor antibody (anti-Tac) and analysis by one- and two-dimensional polyacrylamide gel electrophoresis. The nascent precursor of the receptor (Mr = about 40,000, pI = 6.2-6.5) underwent a post-translational modification giving rise to the mature receptor (IL-2R; Mr = 60,000-65,000, pI = 4.2-4.7) within 2-4 hr. The post-translational processing of IL-2R caused a 20,000-25,000 increase in apparent molecular weight and a 2.0-2.5 acidic shift in the isoelectric point. The increase in molecular weight was attributable mainly to addition of sugar residues including glucosamine and galactose, and the charge shift to the addition of sialic acids. A carboxylic ionophore monensin completely blocked the maturation of IL-2R at the mid-stage of the processing. Fatty acid attachment appeared to comprise one of the steps of the post-translational modification. Two-dimensional analyses of IL-2R biosynthesis enabled identification of the precursor of IL-2R and its intermediate forms, from which it was partially possible to estimate reactions involved in the maturation of the precursor molecule.     

Ectoine disperses keratin and alters hydration kinetics in stratum corneum

Moisturizing compounds are commonly applied topically to human stratum corneum (SC). Many types of molecular species are employed, most commonly including humectants and occlusives. We find new evidence of keratin dispersion caused by the moisturizing compound ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid), and provide the first characterization of its impacts on the hydration kinetics and biomechanics of SC. A second compound, 2-(2-hydroxyethoxy)ethylguanidine succinate (HEG) was investigated for comparison. A suite of biomechanical and biochemical assays including FTIR, drying stress, and cellular cohesion were used. Studies were conducted on normal, lipid-extracted, and lipid plus natural moisturizing factor extracted SC. Ectoine was found to improve the dispersity and hydration of keratin bundles in corneocytes. It also decreased rates of stress development in lipid extracted SC when exposed to a dry environment by ～30% while improving stress reduction during rehydration by ～20%. Peak stresses were increased in harsh drying environments of <5% RH, but SC swelling measurements suggest that water retention was improved in ambient conditions. Further, changes up to ～4 J/m² were seen in cohesion after ectoine treatments, suggesting corneodesmosome interactions. HEG was tested and found to disperse keratin without impacting corneodesmosomes. These results indicate that keratin dispersants produce beneficial effects on SC hydration kinetics, ultimately resulting in higher SC hydration under ambient conditions.     

Determination of the molecular weights of ribonuclease isozymes in a cell-free crude extract of Dictyostelium discoideum, by activity-staining of gels after SDS-PAGE.

1. In a previous report we described three isozymes of intracellular ribonuclease in Dictyostelium discoideum, which were found in vegetative cells. Here we report that the molecular weights of the three isozymes from vegetative cells. 2. They are 14.3 kDa, 60 kDa and 80 kDa, as determined by activity-staining of gels after SDS-PAGE. 3. For renaturation of ribonucleolytic activity from D. discoideum cells after SDS-PAGE, fibrinogen-containing gels were used and gels were washed in aqueous isopropanol to remove detergent. Results of studies by this method suggest that each of these isozymes is composed of only a single polypeptide. 4. The effect of the buffer system on this technique is discussed.     

CGAT: a comparative genome analysis tool for visualizing alignments in the analysis of complex evolutionary changes between closely related genomes

Background  

The recent accumulation of closely related genomic sequences provides a valuable resource for the elucidation of the evolutionary histories of various organisms. However, although numerous alignment calculation and visualization tools have been developed to date, the analysis of complex genomic changes, such as large insertions, deletions, inversions, translocations and duplications, still presents certain difficulties.     

Crosscultural differences in frontalis muscle tension levels: an exploratory study comparing Japanese and Westerners

This paper describes a comparison between Japanese (N = 20) and Westerners (N = 20) in their respective frontal EMG levels and their ability to relax during one 5-minute biofeedback training session. The data indicated a significantly lower baseline muscle tension level in Japanese than in Westerners. After EMG biofeedback, however, no significant difference was found between the two groups. Japanese females showed significantly higher tension levels both during baseline and biofeedback phases than did japanese males.     

Nonspecific Elicitation of Antibody-Forming Cells in the Mouse Spleen by Bacterial Lipopolysaccharide

Mechanisms of nonspecific elicitation of anti-sheep erythrocyte (SRBC) hemolytic antibody plaque-forming cells (PFC) in mouse spleens with an injection of bacterial endotoxin (lipopolysaccharide (LPS)) were studied in comparison with the genesis of naturally occurring ‘background’ PFC in normal mouse spleens and of rapidly arising PFC in mouse spleens after immunization with SRBC. The cytokinetic pattern of anti-SRBC PFC response after an injection of LPS was quite different from that of the response elicited after immunization with SRBC. In addition, even though LPS nonspecifically elicited anti-SRBC PFC response in mice, LPS could not confer any immunological memory on mouse immunocytes for a ‘secondary-type’ anti-SRBC PFC response to restimulation with LPS or SRBC. The administration of rabbit anti-mouse thymocyte immunoglobulin or anti-SRBC antiserum in mice markedly suppressed the PFC response after immunization with SRBC, but did not do so after stimulation with LPS. Neonatally thymectomized mice could still respond to stimulation with LPS, producing anti-SRBC PFC in their spleens. Injections of actinomycin D or cyclophosphamide into mice resulted in obvious reductions of the PFC responses elicited by either LPS or SRBC. However, injections of these immunosuppressive antisera or drugs did not affect the number of anti-SRBC PFC in normal mouse spleens. These results suggest that the geneses of anti-SRBC PFC developed under different conditions, i.e., background PFC, LPS-stimulated PFC, and antigen-stimulated PFC, are quite different from each other, and that the nonspecific elicitation of anti-SRBC PFC by LPS does not require the helper function of T lymphocytes. No obvious difference, however, was observed in the time of ontogenic maturation among these three different anti-SRBC PFC in the mouse spleens judging from when they were first manifested after birth.     

Surface and subsurface structures of neuromasts in tadpoles of the crab-eating frog, Rana cancrivora

Neuromast structure in Rana cancrivora larvae was observed by scanning and transmission electron microscopy. Neuromast units, each being composed of two or three neuromasts, are arranged in several well-defined lines in the head, body, and tail regions. The structure of neuromasts in these three regions is basically identical. The neuromast is composed of sensory, sustentacular, and mantle cells. The top of each neuromast has a hillocklike appearance, and is surrounded by four to six epidermal cells with tight intercellular junctions. Long kinocilia and many stereocilia occur in the apex of the neuromasts and are surrounded by numerous microvilli. Numerous granules are present on the apical portions of the mantle and the sustentacular cells. Four or five trapeziform mantle cells are connected closely with each other to form the shell of the neuromast. Large intercellular spaces occur between the mantle cells and the cells of the inner epidermal layers, and between the cells of the inner epidermal layer. Thus, at the apical parts of the neuromast intercellular junctions are tight and the intercellular spaces are more dilated in more basal areas. Morphologically the neuromasts of R. cancrivora larvae resemble those of generalized pond anurans, based on the grouping of Lannoo (Journal of Morphology 191:115-129, 1987a), although larvae of this species inhabit brackish water.     

Blood kinetics of four intraperitoneally administered therapeutic candidate bacteriophages in healthy and neutropenic mice

Due to multiple-drug resistant bacteria, phage therapy is being revisited. Although most animal experiments focus on therapeutic efficacy, the blood clearance kinetics of phages have not been well described. For further development of an efficient therapeutic strategy, information on phage blood kinetics is important. In this study, time-course concentration changes in peripheral blood of healthy and neutropenic mice were measured using four therapeutic phages (φMR11, KPP10, φEF24C, and KEP10). The results showed a two- to three-day rapid phage clearance, which fits a two-compartment model.