Unraveling tissue regeneration pathways using chemical genetics

Identifying the molecular pathways that are required for regeneration remains one of the great challenges of regenerative medicine. Although genetic mutations have been useful for identifying some molecular pathways, small molecule probes of regenerative pathways might offer some advantages, including the ability to disrupt pathway function with precise temporal control. However, a vertebrate regeneration model amenable to rapid throughput small molecule screening is not currently available. We report here the development of a zebrafish early life stage fin regeneration model and its use in screening for small molecules that modulate tissue regeneration. By screening 2000 biologically active small molecules, we identified 17 that specifically inhibited regeneration. These compounds include a cluster of glucocorticoids, and we demonstrate that transient activation of the glucocorticoid receptor is sufficient to block regeneration, but only if activation occurs during wound healing/blastema formation. In addition, knockdown of the glucocorticoid receptor restores regenerative capability to nonregenerative, glucocorticoid-exposed zebrafish. To test whether the classical anti-inflammatory action of glucocorticoids is responsible for blocking regeneration, we prevented acute inflammation following amputation by antisense repression of the Pu.1 gene. Although loss of Pu.1 prevents the inflammatory response, regeneration is not affected. Collectively, these results indicate that signaling from exogenous glucocorticoids impairs blastema formation and limits regenerative capacity through an acute inflammation-independent mechanism. These studies also demonstrate the feasibility of exploiting chemical genetics to define the pathways that govern vertebrate regeneration.     

Agarose–gelatin conjugate for adherent cell-enclosing capsules

Spherical capsules were prepared by extruding aqueous agarose–gelation conjugate solution into co-flowing liquid paraffin at 38°C and cooling the resultant emulsion. Capsule diameter was controlled between 40 and 250 μm by changing the velocity of the liquid paraffin. Adherent Crandall–Reese feline kidney cells enclosed in conjugate capsules of 141 ± 23 μm diam. had a higher degree of proliferation than those in unmodified agarose capsules. Mitochondrial activity, detected for cell-enclosing conjugate capsules normalized against unit volume of gel, was about double that of unmodified agarose capsules over 28 days. These results demonstrated the feasibility of agarose–gelatin conjugate as a material of cell-enclosing capsules.     

Superoxide dismutase as a target enzyme for Fe-porphyrin-induced cell death

The cell viability of human cancer cell lines treated with [5,10-bis(N-methyl-4-pyridyl)-15,20-diphenyl]porphinatoiron(III) (cis-FeMPy(2)P(2)P) has been estimated. The cis-FeMPy(2)P(2)P is a superoxide dismutase (SOD) mimic in vitro that exhibited a significant toxicity in cancer cell lines. This toxicity is rather due to pro-oxidant properties of the iron-porphyrin in vivo. We have demonstrated that there was the relationship between the LD(50) values calculated from the viability of cancer cell lines treated with cis-FeMPy(2)P(2)P and the SOD activities of the cell lines. Furthermore, the inhibition of SOD by antisense S-oligonucleotide increased the cytotoxic effect of cis-FeMPy(2)P(2)P against cancer cells. These results suggest that SOD is a target enzyme for the cell death induced by cis-FeMPy(2)P(2)P as a new class of anticancer agents.     

Hypervariable loci that enhance the discriminatory ability of newly proposed 15-loci and 24-loci variable-number tandem repeat typing method on Mycobacterium tuberculosis strains predominated by the Beijing family

The newly proposed 15- and 24-loci mycobacterial interspersed repetitive unit (MIRU)-variable-number tandem repeat (VNTR) typing method was evaluated for its ability to differentiate 181 Mycobacterium tuberculosis Beijing family strains. Compared with the original 12-loci MIRU-VNTR typing method, the 15-loci system dramatically improved the discriminatory power for Beijing strains; however, large clusters that could be further differentiated by IS6110 restriction fragment length polymorphism (RFLP) were still obtained. The clonal stability and allelic diversity of a total of 31 VNTR loci were evaluated. VNTRs 3232, 3820, and 4120 were identified as the effective hypervariable VNTR set for the second-line typing of clustered strains following the 15-loci based scheme. Consequently, the discriminatory power of the new scheme (18 loci) equaled that of IS6110 RFLP.     

Distribution of leptocephali of the freshwater eels,genus <Emphasis Type="Italic">Anguilla</Emphasis>, in the waters off west Sumatra in the Indian Ocean

A research cruise was conducted in the eastern Indian Ocean off west Sumatra, Indonesia, in June 2003 to learn about the spawning and larval ecology of the tropical freshwater eels of the genus Anguilla in the region. A total of 43 anguillid leptocephali were collected during the cruise and they were genetically identified as 41 Anguilla bicolor bicolor, 1 Anguilla marmorata, and 1 Anguilla interioris. A. bicolor bicolor leptocephali were 44.1–55.5 mm TL and most of them were at the fully grown stage. Reexamination of the historical data of Jespersen (1942) also suggested a relatively low abundance of small size leptocephali (<40 mm) of this species off west Sumatra. Although the study area has long been considered to be a spawning site of A. bicolor bicolor, the distributions of leptocephali from the two surveys and the patterns of ocean currents in the region suggest the possibility that the main spawning area of this species is located farther offshore.     

Serotypes, antimicrobial susceptibility, and gyr A gene mutation of Campylobacter jejuni isolates from humans and chickens in Thailand

In Thailand, 51% (36/70) Campylobacter jejuni isolates from humans and 68% (47/69) isolates from poultry were classified into 10 Penner serotypes (serotype B, C, R, E, G, A, K, D, I, and L) and 9 serotypes (serotype A, C, I, K, B, E, S, D, and L), respectively. The rate of antimicrobial drug resistance to nalidixic acid, ciprofloxacin, ampicillin, tetracycline, and erythromycin shown by human isolates were 96%, 96%, 29%, 57%, and 14%, while that shown by poultry isolates were 77%, 77%, 22%, 26%, and 17%, respectively. All quinolone-resistant strains contained a mutation in the gyrA gene (T(86)-->I(86)), suggesting that the strains were already widespread in Thailand.     

Molecular cloning and characterization of an arginine decarboxylase gene up-regulated by chilling stress in rice seedlings

We cloned a rice cDNA encoding a putative arginine decarboxylase (ADC) protein, a key enzyme involved with putrescine (Put) biosynthesis in plants. The isolated full-length cDNA (OsADC1) contains an insert consisting of 2451 bp. The longest open reading frame within encodes a putative protein of 702 amino acids, with a calculated molecular mass of 74 kDa and an isoelectric point of 4.9. ClustalW alignment revealed that the deduced OsADC1 protein sequence shares overall 60% and 61% identity at the amino acid level with the Pisum sativum and Glycine max ADC proteins, respectively. Additionally, several OsADC1 regions exhibited striking similarity with these two other plant ADC protein sequences, including motifs characteristic of ADC proteins. Further, RNA gel blot analysis revealed markedly increased OsADC1 mRNA levels in rice seedling leaves subjected to chilling stress. Interestingly, this treatment induced a concomitant increase in free Put levels in these samples, coincident with the observed elevated OsADC1 mRNA levels. To our knowledge, this represents the first direct evidence supporting essentially chilling-specific regulation of a rice ADC gene that also potentially influences Put accumulation, a phenomenon previously noted in cold-stressed rice seedlings.