Antitumor effect of photodynamic therapy with zincphyrin, zinc-coproporphyrin III, in mice

We studied the antitumor effects of photodynamic therapy (PDT) with Zincphyrin, coproporphyrin III with zinc, derived from Streptomyces sp. AC8007, in vitro and in vivo. The photokilling effect of Zincphyrin in the presence of 0.78-100 microg/ml with visible light of 27.2 mW x min/cm2 for 10 min was lower than the hematoporphyrin (Hp) used as a control with L5178Y or sarcoma-180 cells. On the other hand, Zincphyrin apparently reduced tumor growth after intraperitoneal injection at doses of 12.5-50 mg/kg with light irradiation of 75.48 mW x min/cm2 for 10 min in sarcoma-180-bearing mice. Although no mice treated with Zincphyrin died, Hp did cause the death of mice. In B-16 melanoma-bearing mice, both Zincphyrin and Hp had a similar phototherapic effect. Further improvement of the phototherapic effect was observed with the continuous administration of Zincphyrin at 12.5 mg/kg per day for 3 days. The concentration of Zincphyrin in the serum reached a maximum level of 16 microg/ml within 20 min, and the concentration remained at 4.2 microg/ml at 1 hour after the onset of treatment, indicating its rapid action in the body. No animals died after the intraperitoneal administration of Zincphyrin at 100 mg/kg plus exposure to light of 10 mW x min/cm2 for 2 hours, and the body weight of the mice did not decrease. In contrast, all animals receiving 100 mg/kg of Hp under the same conditions died. These results indicate that Zincphyrin would be a useful photosensitizer with low phototoxicity.     

A Multicenter,Randomized, Double-Blind,Placebo-Controlled Trial of High-Dose Rebamipide Treatment for Low-Dose Aspirin-Induced Moderate-to-Severe Small Intestinal Damage

Background

Low-dose aspirin (LDA) frequently causes small bowel injury. While some drugs have been reported to be effective in treating LDA-induced small intestinal damage, most studies did not exclude patients with mild damage thought to be clinically insignificant.

Aim

We conducted a multicenter, randomized, double-blind, placebo-controlled trial to assess the efficacy of a high dose of rebamipide, a gastroprotective drug, for LDA-induced moderate-to-severe enteropathy.

Methods

We enrolled patients who received 100 mg of enteric-coated aspirin daily for more than 3 months and were found to have more than 3 mucosal breaks (i.e., erosions or ulcers) in the small intestine by capsule endoscopy. Eligible patients were assigned to receive either rebamipide 300 mg (triple dose) 3 times daily or placebo for 8 weeks in a 2:1 ratio. Capsule endoscopy was then repeated. The primary endpoint was the change in the number of mucosal breaks from baseline to 8 weeks. Secondary endpoints included the complete healing of mucosal breaks at 8 weeks and the change in Lewis score (an endoscopic score assessing damage severity) from baseline to 8 weeks.

Results

The study was completed by 38 patients (rebamipide group: n = 25, placebo group: n = 13). After 8 weeks of treatment, rebamipide, but not placebo, significantly decreased the number of mucosal breaks (p = 0.046). While the difference was not significant (p = 0.13), the rate of complete mucosal break healing in the rebamipide group (32%, 8 of 25) tended to be higher than that in the placebo group (7.7%, 1 of 13). Rebamipide treatment significantly improved intestinal damage severity as assessed by the Lewis score (p = 0.02), whereas placebo did not. The triple dose of rebamipide was well tolerated.

Conclusions

High-dose rebamipide is effective for the treatment of LDA-induced moderate-to-severe enteropathy.

Trial Registration

UMIN Clinical Trials Registry UMIN000003463     

Conditions Favorable for the Somatic Embryogenesis in Carrot Cell Culture Enhance Expression of the roIC Promoter-GUS Fusion Gene

We obtained carrot (Daucus carota) cells possessing the 5′-noncoding sequence of the ORF12 gene (roIC) of TL-DNA of the Ri plasmid and a structural gene of bacterial β-glucuronidase by Agrobacterium-mediated transformation. When such cells were cultured in medium containing 2,4-dichlorophenoxyacetic acid, substantial reduction in β-glucuronidase activity was observed. Upon transferring the cells from a 2,4-D-containing medium to one devoid of 2,4-dichlorophenoxyacetic acid, enhanced expression of β-glucuronidase in somatic embryo development was recorded. Activation by gibberillic acid and suppression by abscisic acid of β-glucuronidase activities, in concord with embryogenesis, were also noted.     

Fmoc-based solid-phase synthesis of GPR54-agonistic pentapeptide derivatives containing alkene- and fluoroalkene-dipeptide isosteres

Fmoc-protected Phe-Gly-type (Z)-alkene dipeptide isostere (ADI) and (E)-fluoroalkene dipeptide isostere (FADI) were synthesized and applied to Fmoc-based solid-phase peptide synthesis (SPPS). These cis-peptide bond mimetics were introduced into a bioactive pentapeptide [H-Amb-Phe-Gly-Leu-Arg-Trp-NH(2); Amb = 4-(aminomethyl) benzoic acid], which has potent GPR54 agonistic activity. The resulting pentapeptide derivatives showed low GPR54 agonistic activity, as compared with the parent peptide and (E)-ADI-containing derivative. This suggests that the trans-amide conformer of Phe-Gly peptide bond of the parent peptide would be significantly important for bioactivity. Contrary to our expectations, a (Z)-FADI-containing derivative exhibited essentially no activity, revealing the necessity of critical validation of FADI-bioisosterism.     

Rho kinase inhibitors stimulate the migration of human cultured osteoblastic cells by regulating actomyosin activity

We investigated the effects of Rho-associated kinase (ROCK) on migration and cytoskeletal organization in primary human osteoblasts and Saos-2 human osteosarcoma cells. Both cell types were exposed to two different ROCK inhibitors, Y-27632 and HA-1077. In the improved motility assay used in the present study, Y-27632 and HA-1077 significantly increased the migration of both osteoblasts and osteosarcoma cells on plastic in a dose-dependent and reversible manner. Fluorescent images showed that cells of both types cultured with Y-27632 or HA-1077 exhibited a stellate appearance, with poor assembly of stress fibers and focal contacts. Western blotting showed that ROCK inhibitors reduced myosin light chain (MLC) phosphorylation within 5 min without affecting overall myosin light-chain protein levels. Inhibition of ROCK activity is thought to enhance the migration of human osteoblasts through reorganization of the actin cytoskeleton and regulation of myosin activity. ROCK inhibitors may be potentially useful as anabolic agents to enhance the biocompatibility of bone and joint prostheses.     

Growth factor combination for chondrogenic induction from human mesenchymal stem cell

During the last decade, many strategies for cartilage engineering have been emerging. Stem cell induction is one of the possible approaches for cartilage engineering. The mesenchymal stem cells (MSCs) with their pluripotency and availability have been demonstrated to be an attractive cell source. It needs the stimulation with cell growth factors to make the multipluripotent MSCs differentiate into chondrogenic lineage. We have shown particular patterns of in vitro chondrogenesis induction on human bone marrow MSCs (hBMSCs) by cycling the growth factors. The pellet cultures of hBMSCs were prepared for chondrogenic induction. Growth factors: TGF-beta3, BMP-6, and IGF-1 were used in combination for cell induction. Gene expression, histology, immunohistology, and real-time PCR methods were measured on days 21 after cell induction. As shown by histology and immunohistology, the induced cells have shown the feature of chondrocytes in their morphology and extracellular matrix in both inducing patterns of combination and cycling induction. Moreover, the real-time PCR assay has shown the expression of gene markers of chondrogenesis, collagen type II and aggrecan. This study has demonstrated that cartilage tissue can be created from bone marrow mesenchymal stem cells. Interestingly, the combined growth factors TGF-beta3 and BMP-6 or TGF-beta3 and IGF-1 were more effective for chondrogenesis induction as shown by the real-time PCR assay. The combination of these growth factors may be the important key for in vitro chondrogenesis induction.     

Gastrulation in the sea urchin embryo: a model system for analyzing the morphogenesis of a monolayered epithelium

Processes of gastrulation in the sea urchin embryo have been intensively studied to reveal the mechanisms involved in the invagination of a monolayered epithelium. It is widely accepted that the invagination proceeds in two steps (primary and secondary invagination) until the archenteron reaches the apical plate, and that the constituent cells of the resulting archenteron are exclusively derived from the veg2 tier of blastomeres formed at the 60-cell stage. However, recent studies have shown that the recruitment of the archenteron cells lasts as late as the late prism stage, and some descendants of veg1 blastomeres are also recruited into the archenteron. In this review, we first illustrate the current outline of sea urchin gastrulation. Second, several factors, such as cytoskeletons, cell contact and extracellular matrix, will be discussed in relation to the cellular and mechanical basis of gastrulation. Third, differences in the manner of gastrulation among sea urchin species will be described; in some species, the archenteron does not elongate stepwise but continuously. In those embryos, bottle cells are scarcely observed, and the archenteron cells are not rearranged during invagination unlike in typical sea urchins. Attention will be also paid to some other factors, such as the turgor pressure of blastocoele and the force generated by blastocoele wall. These factors, in spite of their significance, have been neglected in the analysis of sea urchin gastrulation. Lastly, we will discuss how behavior of pigment cells defines the manner of gastrulation, because pigment cells recently turned out to be the bottle cells that trigger the initial inward bending of the vegetal plate.     

Identification of a yolk sac cell population with hematopoietic activity in view of CD45/c-Kit expression

During murine embryonic development, primitive hematopoiesis occurs in the yolk sac (YS). Recent studies have shown that the YS also harbors definitive hematopoietic activity. However, the population of YS cells contributing to definitive hematopoiesis has not been identified. In this study, we characterized the hematopoietic cell populations in the YS of mouse embryos from E9.5 to E14.5 in view of the expression profiles of CD45 and c-Kit. The YS cells from E9.5 to E11.5 could be divided into six populations: CD45(-) c-Kit(-) , CD45(-) c-Kit(low) , CD45(-) c-Kit(high) , CD45(low) c-Kit(high) , CD45(high) c-Kit(high) and CD45(high) c-Kit(very low) . Among these populations, CD45(low) c-Kit(high) cells showed the highest multilineage hematopoietic colony-forming activity. Later in development, the YS cells from E12.5 to E14.5 lost the second and fourth populations (i.e., they retained CD45(-) c-Kit(-) , CD45(-) c-Kit(high) , CD45(high) c-Kit(high) and CD45(high) c-Kit(very low) cells), and concurrently with the disappearance of the CD45(low) c-Kit(high) population, no significant hematopoietic activity was found in any of the populations on and after E12.5. CD45(low) c-Kit(high) YS cells, which had a round morphology with a large nucleus, possessed the ability to differentiate into myeloid and B lymphoid cells when cultured with stromal cells. These findings suggest that CD45(low) c-Kit(high) YS cells include more undifferentiated cells than the other YS cell populations and possess in vitro potency to differentiate into multilineage hematopoietic cells. Furthermore, this cell population disappears from the YS at around E12.5, when the site of hematopoiesis has already shifted to the fetal liver and the placenta.     

Selection of a carbohydrate-binding domain with a helix-loop-helix structure

We obtained a novel carbohydrate-binding peptide having a helix-loop-helix scaffold from a random peptide library. The helix-loop-helix peptide library randomized at five amino acid residues was displayed on the major coat protein of a filamentous phage. Affinity selection with a ganglioside, Galbeta1-3GalNAcbeta1-4(Neu5Acalpha2-3)Galbeta1-4Glcbeta1-1'Cer (GM1), gave positive phage clones. Surface plasmon resonance spectroscopy showed that a corresponding 35-mer synthetic peptide had high affinity for GM1 with a dissociation constant of 0.24 microM. This peptide preferentially binds to GM1 rather than asialo GM1 and GM2, suggesting that a terminal galactose and sialic acid are required for the binding as for cholera toxin. Circular dichroism spectroscopic studies indicated that a helical structure is important for the affinity and specificity. Furthermore, alanine scanning at randomized positions showed that arginine and phenylalanine play an especially important role in the recognition of carbohydrates. Such a de novo helix-loop-helix peptide would be available for the design of carbohydrate-binding proteins.