Mature and juvenile tissue models of regeneration in small fish species

The multitude of cells constituting organisms are fragile and easily damaged day by day. Therefore, maintenance of tissue morphology and function is fundamental for multicellular organisms to attain long life. For proper maintenance of tissue integrity, organisms must have mechanisms that detect the loss of tissue mass, activate the de novo production of cells, and organize those cells into functional tissues. However, these processes are only poorly understood. Here we give an overview of adult and juvenile tissue regeneration models in small fish species, such as zebrafish and medaka, and highlight recent advances at the molecular level. From these advances, we have come to realize that the epidermal and mesenchymal parts of the regenerating fish fin-that is, the wound epidermis and blastema, respectively-comprise heterogeneous populations of cells with different molecular identities that can be termed "compartments." These compartments and their mutual interactions are thought to play important roles in promoting the proper progression of tissue regeneration. We further describe the current understanding of these compartments and discuss the possible approaches to affording a better understanding of their roles and interactions during regeneration.     

Heme-dependent activation of neuronal nitric oxide synthase by cytosol is due to an Hsp70-dependent, thioredoxin-mediated thiol-disulfide interchange in the heme/substrate binding cleft

We have reported that heme-dependent activation of apo-neuronal nitric oxide synthase (apo-nNOS) to the active holo-enzyme dimer is dependent upon factors present in reticulocyte lysate and other cytosols. Here, we find that both Hsp70 and thioredoxin are components of the activation system. The apo-nNOS activating activity of reticulocyte lysate is retained in a pool of fractions containing Hsp70 that elute from DE52 prior to Hsp90. All of the activating activity and 20-30% of the Hsp70 elute in the flow-through fraction upon subsequent ATP-agarose chromatography. Apo-nNOS activation by this flow-through fraction is inhibited by pifithrin-μ, a small molecule inhibitor of Hsp70, suggesting that a non-ATP-binding form of Hsp70 is involved in heme-dependent apo-nNOS activation. Previous work has shown that apo-nNOS can be activated by thiol-disulfide exchange, and we show substantial activation with a small molecule dithiol modeled on the active motifs of thioredoxin and protein disulfide isomerase. Further fractionation of the ATP-agarose flow-through on Sephacryl S-300 separates free thioredoxin from apo-nNOS activating activity, Hsp70, and a small amount of thioredoxin, all of which are eluted throughout the macromolecular peak. Incubation of apo-nNOS with the macromolecular fraction in combination either with the thioredoxin-containing fraction or with purified recombinant human thioredoxin restores full heme-dependent activating activity. This supports a model in which Hsp70 binding to apo-nNOS stabilizes an open state of the heme/substrate binding cleft to facilitate thioredoxin access to the active site cysteine that coordinates with heme iron, permitting heme binding and dimerization to the active enzyme.     

Flavone-catalyzed apoptosis in Scutellaria baicalensis

In response to mechanical damage, roots of Scutellaria baicalensis undergo cell death within 24 h. The flavone baicalein was identified as the factor regulating apoptosis in the damaged roots of S. baicalensis. Plant apoptosis is known to be triggered by oxidative damage of DNA through oxidative bursts, whereas baicalein causes apoptosis in Scutellaria cells by a copper-dependent oxidation of nuclear DNA without inducing an oxidative burst. S. baicalensis possesses an interesting system for quickly producing this apoptosis-inducing flavone in its cells. Intact Scutellaria cells contain little baicalein but store a large amount of baicalin (baicalein 7-O-β-D-glucuronide). Stress treatment of Scutellaria cells immediately initiates hydrolysis of baicalin by endogenous β-glucuronidase, and the resulting baicalein is immediately translocated to the nucleus, leading to apoptosis. Thus, S. baicalensis possesses a unique apoptosis-inducing system that is linked with metabolism of baicalin.     

Allosteric drugs: the interaction of antitumor compound MKT-077 with human Hsp70 chaperones

Hsp70 (heat shock protein 70 kDa) chaperones are key to cellular protein homeostasis. However, they also have the ability to inhibit tumor apoptosis and contribute to aberrant accumulation of hyperphosphorylated tau in neuronal cells affected by tauopathies, including Alzheimer's disease. Hence, Hsp70 chaperones are increasingly becoming identified as targets for therapeutic intervention in these widely abundant diseases. Hsp70 proteins are allosteric machines and offer, besides classical active-site targets, also opportunities to target the mechanism of allostery. In this work, it is demonstrated that the action of the potent anticancer compound MKT-077 (1-ethyl-2-[[3-ethyl-5-(3-methylbenzothiazolin-2-yliden)]-4-oxothiazolidin-2-ylidenemethyl] pyridinium chloride) occurs through a differential interaction with Hsp70 allosteric states. MKT-077 is therefore an “allosteric drug.” Using NMR spectroscopy, we identify the compound's binding site on human HSPA8 (Hsc70). The binding pose is obtained from NMR-restrained docking calculations, subsequently scored by molecular-dynamics-based energy and solvation computations. Suggestions for the improvement of the compound's properties are made on the basis of the binding location and pose.     

Novel potent antagonists of human neuropeptide Y Y5 receptors. Part 2: substituted benzo[a]cycloheptene derivatives

Novel benzo[a]cycloheptene derivatives were prepared for the purpose of searching new neuropeptide Y-Y5 (NPY-Y5) receptor antagonists. The structure-activity relationships are described and compound 2o (FR226928) showed the most potent affinity for Y5 receptor of all we prepared and was found to have higher potency and better selectivity for Y5 over Y1 receptor affinities when compared with the known lead compound 1.     

A simple allele-specific PCR marker for identifying male-sterile trees: Towards DNA marker-assisted selection in the Cryptomeria japonica breeding program

The number of people in Japan suffering from Cryptomeria japonica pollinosis has risen considerably since the 1970s as the area planted with this species has increased. In order to reduce the amount of pollen dispersed, breeding programs using trees with male-sterile genes have been implemented. We have constructed partial linkage maps surrounding a male sterility gene (ms-1) in four families of C. japonica to facilitate this process. The marker most closely linked to ms-1 was different in the four mapping families: gSNP00438, gSNP01452, estSNP00083, and estSNP01228 in the TO13S family (3.1 cM from ms-1); gSNP05835 and gSNP06239 in the S3T67 family (2.0 cM from ms-1); gSNP05835 in the F1N4 family (1.5 cM from ms-1); and gSNP06239 in the T5 family (4.2 cM from ms-1). This is probably mainly due to genetic differences between the parents used to produce the mapping families. However, in all four families, the accuracy with which male-sterile trees could be identified using the closest markers was more than 96.0 %. These results suggested that marker-assisted selection of male-sterile trees within a given family is feasible using the closest flanking markers to the ms-1 locus. We also developed an allele-specific PCR marker for identifying male-sterile trees in the TO13S family from which male-sterile seedlings are produced. Allele-specific PCR using three primer combinations produced two clear fragments, which could be easily separated by agarose gel electrophoresis: one fragment with a molecular weight of 410 bp, which was present in all samples and could thus be used as a positive control, and another of lower molecular weight (196 bp), which was specific for male-sterile trees. This marker makes it possible to carry out a simple and economical PCR assay for the detection of the SNP linked to the target gene without the need to use fluorescent labels. This study shows how a simple allele-specific PCR marker for an important major gene in a forest tree species can be developed using information from a high-density linkage map. In addition, our results will facilitate the first application of MAS (marker assisted selection) in conifers because the male sterility in C. japonica has several advantages and may be one of the best examples for MAS in conifers.     

Frequency of and Predictive Factors for Vascular Invasion after Radiofrequency Ablation for Hepatocellular Carcinoma

Background

Vascular invasion in patients with hepatocellular carcinoma (HCC) is representative of advanced disease with an extremely poor prognosis. The detailed course of its development has not been fully elucidated.

Methods

We enrolled 1057 consecutive patients with HCC who had been treated with curative intent by radiofrequency ablation (RFA) as an initial therapy from 1999 to 2008 at our department. We analyzed the incidence rate of and predictive factors for vascular invasion. The survival rate after detection of vascular invasion was also analyzed.

Results

During a mean follow-up period of 4.5 years, 6075 nodules including primary and recurrent lesions were treated by RFA. Vascular invasion was observed in 97 patients. The rate of vascular invasion associated with site of original RFA procedure was 0.66% on a nodule basis. The incidence rates of vascular invasion on a patient basis at 1, 3, and 5 years were 1.1%, 5.9%, and 10.4%, respectively. Univariate analysis revealed that tumor size, tumor number, alpha-fetoprotein (AFP), des-gamma-carboxy prothrombin (DCP), and Lens culinaris agglutinin-reactive fraction of alpha-fetoprotein were significant risk predictors of vascular invasion. In multivariate analysis, DCP was the most significant predictor for vascular invasion (compared with a DCP of ≤100 mAu/mL, the hazard ratio was 1.95 when DCP was 101–200 mAu/mL and 3.22 when DCP was >200 mAu/mL). The median survival time after development of vascular invasion was only 6 months.

Conclusion

Vascular invasion occurs during the clinical course of patients initially treated with curative intent. High-risk patients may be identified using tumor markers.     

Differential patterns of expression of glycosylphosphatidylinositol-anchored carcinoembryonic antigen and alkaline phosphatase in various cancer cell lines

The expression of glycosylphosphatidylinositol (GPI-anchored) carcinoembryonic antigen (CEA) and alkaline phosphatase (ALP) on the cell surface of various cancer cell lines and a lung diploid cell line (WI38) was investigated, with exposure of the cell lines to a cell differentiation agent (sodium butyrate) to induce cell differentiation and expression of the two tumor-associated antigens. In three colon (SW1222, SW1116, and HT-29) and stomach (MKN-45) cancer cell lines, all of which are double producers of CEA and ALP, the maximum expression of GPI-anchored CEA occurred with butyrate at a lower concentration than did that of GPI-anchored ALP. GPI-anchored ALP derived from colon (SW1222 and SW1116) and stomach (MKN-45 and MKN-1) cancer cell lines was heat-stable with and without exposure to butyrate, but GPI-anchored ALP derived from lung cancer cell lines (PC-6, PC13, PC-14, WI26VA4, and WI38VA13) showed a variety of heat stabilities, depending on cell line, butyrate exposure, and SV40 transformation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Relationship between antibody productivity by activated human lymph node lymphocytes from lung cancer patients and lymphocyte subsets

Regional lymph node lymphocytes from five patients with primary lung cancer were analyzed for subset composition, and exposed in vitro to the polyclonal human B cell mitogen Staphylococcus aureus Cowan I (SACI) or the murine B cell mitogen lipopolysaccharide (LPS) and then fused with mouse myeloma cells for investigation at the clonal level of their antibody (Ab) production and its statistical relation to the original subset composition. No correlation was found between the proportion of CD19+, CD23+, or CD3+ cells in the lymphocyte sample prior to its exposure to either SACI or LPS, and the Ab production efficiency, defined as the ratio of the number of Ab producing wells to the total number of proliferating wells. For lymphocytes exposed to LPS, however, a strong correlation (r = 0.931, p = 0.02) was observed between the Ab production efficiency and the ratio of CD8+ to CD3+ cells (CD8/CD3) in the original sample at least within the ranges studied (CD8/CD3 = 0.216–0.288). For those exposed to SACI, no correlation was found between the Ab production efficiency and the CD8/CD3 ratio (r = 0.881, p = 0.12) or the proportion of CD8+ cells (r = 0.808, p = 0.19) in the original sample. These results suggest that the repertoire of B cells responsive to LPS is different at least in part from the repertoire responsive to SACI and that the ratio CD8/CD3 could serve as a practical predictor for Ab production by human lymphocytes stimulated with LPS. This revised version was published online in July 2006 with corrections to the Cover Date.     

DENSITY-DEPENDENT SELECTION ON CONTINUOUS CHARACTERS: A QUANTITATIVE GENETIC MODEL

A quantitative genetic model of density-dependent selection is presented and analysed with parameter values obtained from laboratory selection experiments conducted by Mueller and his coworkers. The ecological concept of r- and K-selection is formulated in terms of selection gradients on underlying phenotypic characters that influence the density-dependent measure of fitness. Hence the selection gradients on traits are decomposed into two components, one that changes in the direction to increase r, and one that changes in the direction to increase K. The relative importance of the two components is determined by temporal fluctuations in population density. The evolutionary rate of r and K (per-generation changes in r and K due to the genetic responses of the underlying traits) is also formulated. Numerical simulation has shown that with moderate genetic variances of the underlying characters, r and K can evolve rapidly and the evolutionary rate is influenced by synergistic interaction between characters that contribute to r and K. But strong r-selection can occur only with severe and continuous disturbances of populations so that the population density is kept low enough to prevent K-selection.