Large-scale analysis of the genes involved in fin regeneration and blastema formation in the medaka, Oryzias latipes

Medaka is an attractive model to study epimorphic regeneration. The fins have remarkable regenerative capacity and are replaced about 14 days after amputation. The formation of blastema, a mass of undifferentiated cells, is essential for regeneration; however, the molecular mechanisms are incompletely defined. To identify the genes required for fin regeneration, especially for blastema formation, we constructed cDNA libraries from fin regenerates at 3 days postamputation and 10 days postamputation. A total of 16,866 expression sequence tags (ESTs) were sequenced and subjected to BLASTX analysis. The result revealed that about 60% of them showed strong matches to previously identified proteins, and major signaling molecules related to development, including FGF, BMP, Wnt, Notch/Delta, and Ephrin/Eph signaling pathways were isolated. To identify novel genes that showed specific expression during fin regeneration, cDNA microarray was generated based on 2900 independent ESTs from each library which had no sequence similarity to known proteins. We obtained 6 candidate genes associated with blastema formation by gene expression pattern screening in competitive hybridization analyses and in situ hybridization. Olrfe16d23 and olrfe14k04 were expressed only in early regenerating stages when blastema formation was induced. The expression of olrf5n23, which encodes a novel signal peptide, was detected in wound epidermis throughout regeneration. Olrfe23l22, olrfe20n22, and olrfe24i02 were expressed notably in the blastema region. Our study has thus identified the gene expression profiles and some novel candidate genes to facilitate elucidation of the molecular mechanisms of fin regeneration.     

Gain- and loss-of-function in chick embryos by electroporation

It remained very difficult to manipulate gene expression in chick embryos until the advent of in ovo electroporation which enabled the induction of both gain-of-function, and recently loss-of-function, of a gene of interest at a specific developmental stage. Gain-of-function by electroporation is so effective that it has become widely adopted in developmental studies in the chick. Recently, it became possible to induce loss-of-function by introducing an siRNA expression vector by electroporation. In this review, the methods of electroporation for gain-of-function and for loss-of-function by siRNA are discussed.     

Thoracic vein ablation terminates chronic atrial fibrillation in dogs

The thoracic vein hypothesis of chronic atrial fibrillation (AF) posits that rapid, repetitive activations from muscle sleeves within thoracic veins underlie the mechanism of sustained AF. If this is so, thoracic vein ablation should terminate sustained AF and prevent its reinduction. Six female mongrel dogs underwent chronic pulmonary vein (PV) pacing at 20 Hz to induce sustained (>48 h) AF. Bipolar electrodes were used to record from the atria and thoracic veins, including the vein of Marshall, four PVs, and the superior vena cava. Radio frequency (RF) application was applied around the PVs and superior vena cava and along the vein of Marshall until electrical activity was eliminated. Computerized mapping (1,792 electrodes, 1 mm resolution) was also performed. Sustained AF was induced in 30.6 +/- 6.5 days, and ablation was done 17.3 +/- 8.5 days afterward. Before ablation, the PVs had shorter activation cycle lengths than the atria, and rapid, repetitive activations were observed in the PVs. All dogs converted to sinus rhythm during (n = 4 dogs) or within 90 min of completion of RF ablation. Rapid atrial pacing afterward induced only nonsustained (<60 s) AF in all dogs. Average AF cycle lengths after reinduction were significantly (P = 0.01) longer (183 +/- 31.5 ms) than baseline (106 +/- 16.2 ms). There were no activation cycle length gradients after RF application. We conclude that thoracic vein ablation converts canine sustained AF into sinus rhythm and prevents the reinduction of sustained AF. These findings suggest that thoracic veins are important in the maintenance of AF in dogs.     

Over-expression of the testis-specific gene TSGA10 in cancers and its immunogenicity

The TSGA10 gene was originally isolated in normal testis by differential mRNA display. TSGA10 is located on chromosome 2q11.2 and consists of 19 exons extending over 3 kb. TSGA10 mRNA expression was investigated in normal and malignant tissues using quantitative real-time RT-PCR. It was predominantly expressed in the testis in adult normal tissues. In malignant tissues, TSGA10 was over-expressed in 4 of 20 hepatocellular carcinomas (HCC), 1 of 20 colon cancers, 7 of 20 ovarian cancers, 3 of 20 prostate cancers, 1 of 21 malignant melanomas, and 8 of 21 bladder cancers. Serological analysis revealed that 3 out of 346 patients with various types of cancer possessed antibody against recombinant TSGA10 protein. They included 2 patients with hepatocellular carcinoma and a patient with malignant melanoma.     

Free dorsoulnar perforator flap transfers for the reconstruction of severely injured digits

The aim of this study was to investigate the feasibility of transferring the free dorsoulnar perforator flap nourished by the cutaneous perforator branched dorsoulnar artery to reconstruct severely injured fingers under upper arm anesthesia. Between April of 2001 and April of 2002, 13 free dorsoulnar perforator flaps were used in 13 patients. There were 11 men and two women ranging in age from 18 to 64 years, with an average age of 38 years. The affected fingers were one thumb, four index fingers, five middle fingers, two ring fingers, and one little finger. All cases were performed under upper arm anesthesia combined with intravenous local anesthesia. The operative time ranged from 103 to 140 minutes, with an average time of 120 minutes. The flap size ranged from 1 x 3 to 3 x 4 cm, and was transferred from the same forearm of the injured finger. All donor sites were closed primarily without a skin graft. The aim of reconstruction for fingers was to repair a traumatic defect (five cases), partial necrosis following replantation (two cases), and soft-tissue defects resulting from resection of a scar (three cases) and to revascularize ischemic fingers (three cases). All flaps survived completely. After repair of the flow-through circulation of the common digital artery and ischemic finger, a postoperative angiogram showed the vascular patency and hypervascularity of the reconstructed fingers, and the patients' complaints were reduced. The free dorsoulnar perforator flap under regional anesthesia is first reported; it may become one valuable option as a very small flap for the treatment of repairing intercalated or segmental defects as a flow-through flap for soft-tissue defects and ischemic fingers.     

Expression of cys-cys chemokine ligand 21 on human gingival lymphatic vessels

The cys-cys (C-C) chemokine ligand 21 is a member of the C-C chemokines that constitute a group of heparin-binding cytokines with a pattern of four or six conserved cysteines. The CCL21 is known to be expressed in secondary lymphoid tissues, however it has rarely been reported for the expression on peripheral lymphatic vessels in somatic tissue. Here we investigated the expression of CCL21 on lymphatic vessels identified by anti-desmoplakin in uninflamed and inflamed human gingiva. In uninflamed tissue the expression of CCL21 was detected on lymphatic vessels in gingiva. In uninflamed gingiva the expression of CCL21 was detected on all lymphatic capillaries of the mucosal connective tissue papillae. There were two types of collecting lymphatic vessels in the lamina propria mucosae expressing CCL21 strongly or very weakly. In inflamed gingiva no expression of CCL21 was detected on lymphatic vessels. In all tissue sections no blood vessels expressing CCL21 were observed. These results may suggest that the expression of CCL21 is predominantly induced in the peripheral lymphatic endothelium of the uninflamed mucosal microcirculation, and that under inflamed conditions a reduction of CCL21 occurs in lymphatic endothelium.     

Identification and isolation of anaerobic, syntrophic phthalate isomer-degrading microbes from methanogenic sludges treating wastewater from terephthalate manufacturing

The microbial populations responsible for anaerobic degradation of phthalate isomers were investigated by enrichment and isolation of those microbes from anaerobic sludge treating wastewater from the manufacturing of terephthalic acid. Primary enrichments were made with each of three phthalate isomers (ortho-, iso-, and terephthalate) as the sole energy source at 37 degrees C with two sources of anaerobic sludge (both had been used to treat wastewater containing high concentrations of phthalate isomers) as the inoculum. Six methanogenic enrichment cultures were obtained which not only degraded the isomer used for the enrichment but also had the potential to degrade part of other phthalate isomers as well as benzoate with concomitant production of methane, presumably involving strictly syntrophic substrate degradation. Our 16S rRNA gene-cloning analysis combined with fluorescence in situ hybridization revealed that the predominant bacteria in the enrichment cultures were affiliated with a recently recognized non-sulfate-reducing subcluster (subcluster Ih) in the group 'Desulfotomaculum lineage I' or a clone cluster (group TA) in the class delta-PROTEOBACTERIA: Several attempts were made to isolate these microbes, resulting in the isolation of a terephthalate-degrading bacterium, designated strain JT, in pure culture. A coculture of the strain with the hydrogenotrophic methanogen Methanospirillum hungatei converted terephthalate to acetate and methane within 3 months of incubation, whereas strain JT could not degrade terephthalate in pure culture. During the degradation of terephthalate, a small amount of benzoate was transiently accumulated as an intermediate, indicative of decarboxylation of terephthalate to benzoate as the initial step of the degradation. 16S rRNA gene sequence analysis revealed that the strain was a member of subcluster Ih of the group 'Desulfotomaculum lineage I', but it was only distantly related to other known species.     

Strategies for bioremediation of polychlorinated biphenyls

Polychlorinated biphenyls (PCBs) are serious environmental pollutants that threaten both the natural ecosystem and human health. For remediation of environments contaminated with PCBs, several approaches that exploit the potential of microbes to degrade PCBs have been developed. These approaches include improvement of PCB solubilization and entry into the cell, pathway and enzyme engineering, and control of enzyme expression. In this mini-review, we briefly summarize these strategies and provide potentially useful knowledge for the further improvement of the bacterial breakdown of PCBs.