A set of polymorphic trinucleotide and tetranucleotide microsatellite markers for silver crucian carp (Carassius auratus gibelio) and cross-amplification in crucian carp.

Silver crucian carp (Carassius auratus gibelio Bloch), as a gynogenetic fish, is a promising model for the study of the evolutionary genetics of vertebrates. We have developed 59 polymorphic trinucleotide and tetranucleotide markers for the silver crucian carp through the biotin capture method and radioactive-labeling hybridization. The number of alleles ranged from 2 to 12 in the population, and the average proportion of heterozygotes (including tri- and diallelic) at polymorphic loci was 76.8%. In addition, these loci were successfully applied to a close relative, the crucian carp (Carassius auratus), by cross-amplification, as shown by the range of alleles (2-19), observed heterozygosity (0.1765-0.9706), expected heterozygosity (0.2392-0.9421), and polymorphism information content (0.2186-0.9236).

[Experimental study on repair of peripheral nerve defect by basic fibroblast growth factor combined with autogenous vein graft conduit].

OBJECTIVE: To explore the effect of basic fibroblast growth factor (bFGF) combined with autogenous vein graft conduit on peripheral nerve regeneration. METHODS: Fifty four New Zealand rabbits were divided into three groups. The main trunk of sciatic nerve of rabbit in one side was severed and bridged by autogenous vein. 0.2 ml bFGF solution (4,000 U/ml) was intravenously injected to the vein graft conduit as group A, the same amount of saline solution as group B, and no solution injection as group C. Microscopic examination, axon video analysis and nerve conduct velocity were performed at the 10th, 30th, and 100th day after operation. RESULTS: The nerve fibers were grown into vein graft conduit in all groups at 30th after operation, they were more and regular in group A than that of group B and C, and the axon regeneration rate in group A was more than that of group B and C. CONCLUSION: bFGF combined with autogenous vein graft conduit can markedly promote nerve regeneration.

Delayed cytotoxicity of 6-mercaptopurine is compatible with mitotic death caused by DNA damage due to incorporation of 6-thioguanine into DNA as 6-thioguanine nucleotide.

Many protocol studies have shown that low dose 6-mercaptopurine (6MP) in maintenance chemotherapy for childhood acute lymphoblastic leukemia (ALL) can be utilized to cure the disease. Mitotic or reproductive cell death has been recognized after G2 arrest when cells are treated with antitumor agents. The precise mechanism of mode of action of 6MP still remains unclear. We found delayed cytotoxic effect of 6MP in P388 murine leukemic cells. Morphological study showed that 6MP induced delayed death was characterized by an enlargement of cell size and multinucleated nuclei. Agarose gel electrophoresis of fragmented DNA from cells treated with 6MP showed the typical ladder pattern. These findings were compatible with mitotic death. Our results make us hypothesize that the delayed cytotoxicity of 6MP is one of the drug induced mitotic deaths caused by DNA damage due to incorporation of 6-thioguanine (6TG) into DNA as thioguanine nucleotide (TGN). Mitotic death may be a mechanism for killing the cycling cells from residual leukemic cells in G0 or long G1 phases in the treatment of childhood ALL.

Enhanced cytotoxic interaction between 5-fluorouracil and 4-hydroperoxycyclophosphamide against L1210 murine leukemic cells: applicability to ex vivo purging.

Eradication of contaminated tumor cells in bone marrow is a matter of utmost concern in the setting of autologous bone marrow transplantation. 4-Hydroperoxycyclophosphamide (4-HC) is often used for ex vivo chemical purging of contaminated tumor cells in bone marrow. The marrow from patients pretreated with 5-fluorouracil (5-FU) is enriched with multifactor-responsive high proliferative potential colony-forming cells. To develop an efficient ex vivo chemical purging system, we evaluated interaction between 4-HC and 5-FU. We investigated the antitumor effect of cyclophosphamide, a mother compound of 4-HC, and 5-FU against L1210 ascites tumor in B6D2F1 mice. The median lifespan of the mice treated with 4-HC or 5-FU alone was 8 and 12 days, respectively. The combination of both drugs significantly extended the median lifespan to 18.5 days. The median effect plot analysis indicated a synergistic cytotoxic interaction between 5-FU and 4-HC in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl terazolium bromide (MTT) assay. Clonogenic assay also showed that combination of 4-HC and 5-FU significantly reduced L1210 leukemic colonies to 20% of untreated control. Bone marrow cells from the mice treated with 5-FU at 150 mg/kg body weight was resistant to 4-HC at concentrations as high as 0.2 microgram/ml, which was more than 70% inhibitory concentration for colony formation in L1210 leukemic cells. Findings suggest that sequential treatment with in vivo 5-FU followed by ex vivo 4-HC could selectively enhance antitumor effects of 4-HC in tumor cells remaining in bone marrow.

Aclarubicin inhibits etoposide induced apoptosis through inhibition of RNA synthesis in P388 murine leukemic cells.

It has been reported that aclarubicin inhibits etoposide (VP-16) induced cytotoxicity in human lung cancer cell lines (1, 2). However, it still remains unclear how aclarubicin (ACR) inhibits etoposide-induced cytotoxicity. We report here that the combination of ACR and VP-16 showed antagonistic cytotoxic effect in P388 murine leukemic cells. DNA unwinding assay showed that 1000 ng/ml ACR significantly reduced VP-16 induced early DNA double strand(ds) breaks compared to that of VP-16 alone at a concentration of 10 microM. However, ACR did not inhibit VP-16 induced early DNA double strand breaks at a concentration of 100 ng/ml, a clinically achievable concentration. Furthermore, DNA repair occurred within two hours after removing VP-16 even if ACR was co-cultured at concentrations of 100 and 1000 ng/ml. DNA agarose gel electrophoresis and detection of sub-G1 fraction by flowcytometer showed that 100 ng/ml of ACR inhibited VP-16 induced DNA ladder formation and formation of sub-G1 fraction. Radioactive precursor incorporation studies showed that VP-16 inhibited DNA synthesis rather than RNA synthesis. On the other hand, ACR selectively inhibited RNA synthesis at a concentration of 100 ng/ml. The VP-16 induced increment of [3H]-L-leucine uptake was canceled by addition of 100 ng/ml of ACR. These data suggest that ACR inhibited VP-16 induced apoptosis by the inhibition of RNA synthesis along with protein synthesis, but not early DNA double strand breaks and DNA repair at a concentration of 100 ng/ml in P388 murine leukemic cells.

Deoxycytidine kinase mRNA levels in leukemia cells with competitive polymerase chain reaction assay.

Deoxycytidine kinase (dCyd kinase) is important for the phosphorylation of several different nucleoside antimetabolites. To understand the significance of dCyd kinase levels in chemotherapy, dCyd kinase mRNA levels were evaluated in several cells with a quantitative competitive polymerase chain reaction (PCR) assay. dCyd kinase catalytic activity and intracellular ara-CTP production were also compared with the levels of dCyd kinase mRNA. The assay was able to show: (i) that dCyd kinase catalytic activity and dCyd kinase mRNA levels were correlated in cells; (ii) that dCyd kinase mRNA levels were more sensitive in lower levels of 10 amol/micrograms of total RNA; and (iii) in cytosine arabinoside (ara-C)-resistant cells, both dCyd kinase mRNA levels and intracellular ara-CTP levels were lower compared with levels in sensitive cells. The PCR assay for dCyd kinase mRNA could be useful in the selection and monitoring of patients treated with nucleosides that are activated by this enzyme.

Heterogeneous effects of G-CSF and GM-CSF on cell growth and ara-C cytotoxicity in childhood leukemias which express myeloid markers.

It is uncertain if acute lymphoblastic leukemia (ALL) cells expressing myeloid makers can respond to granulocyte colony-stimulating factor (G-CSF) and granulocyte macrophage colony-stimulating factor (GM-CSF). We investigated the effects of G-CSF (0.01 microgram/ml) and GM-CSF (0.01 microgram/ml) on [3H]thymidine (TdR) uptake, and the cytotoxicity of 1-beta-D-arabinofuranosylcytosine (ara-C) in leukemia cells from 17 pediatric patients. ALL cells without myeloid markers did not respond to G-CSF or GM-CSF. On the other hand, these cytokines enhanced the [3H]TdR uptake and cell growth, not only of AML cells but also of ALL cells expressing myeloid antigens. However, G-CSF and GM-CSF did not always enhance the growth inhibitory effect of the cell cycle specific drug ara-C when the cells were co-cultured with the drug. There was no relationship between cell growth and the amount of [3H]TdR incorporation or the intracellular ara-CTP level. These results indicate the heterogeneous effects of G-CSF and GM-CSF on cell growth and ara-C sensitivity in childhood leukemia cells.

Combined oral administration of etoposide and arabinofuranosylcytosine-5'-stearylphosphate enhances the antitumor effect against P388 ascites tumors.

We investigated the antitumor effect of oral administration of etoposide and arabinofuranosylcytosine-5'-stearylphosphate (C18PCA) against P388 ascites tumors in B6D2F1 mice. Etoposide (25 mg/kg) and C18PCA (5 mg/kg) were given orally on days 1-5 after tumor inoculation. The median life span of the mice treated with etoposide or C18PCA alone was 19.5 and 18 days, respectively. The combination of both drugs significantly extended the median life span to 33 days. To clarify this enhancement of the increase in median life span, we examined intracellular deoxyribonucleoside triphosphate (dNTP) pools, cell-cycle distribution, DNA fragmentation, and the time course of the plasma drug concentration. Etoposide had no effect on intracellular dNTP pools in this experimental system, whereas treatment of cells with C18PCA or with the combination of both drugs resulted in a significant increase in dTTP pools to values ranging from 1.8- to 2.0-fold higher than the control levels. There was a significant increase in cells in the S + G2/M phase when cells had been treated with both etoposide and C18PCA. Agarose-gel electrophoresis of the extracted DNA revealed that C18PCA enhanced the fragmentation of DNA, with a length of about 180 bp being induced by etoposide. The plasma peak levels of etoposide (1000 nM) and ara-C (50 nM) were observed at 20 and 30 min after the simultaneous administration of both drugs, respectively. The plasma etoposide level gradually decreased to 10% of the peak level at 240 min after administration. On the other hand, the plasma concentration of ara-C was maintained at above 20 nM at 240 min. These observations suggest that C18PCA and etoposide act on P388 murine leukemic cells by accumulating cells in the S + G2/M phase. Even if the plasma concentration of ara-C is low, the repair of DNA damage by etoposide may be hindered in the presence of ara-C following an increase in DNA fragmentation.

Bone marrow transplantation for leukemia after conditioning with busulfan and cyclophosphamide. Bone marrow morphology in the early recovery phase.

The bone marrow morphology of fifty patients with leukemia was analyzed before and two weeks after allogeneic bone marrow transplantation and compared to that of ten normal, healthy control subjects. Conditioning of the marrow graft recipients had been carried out with a combination of 16 mg/kg of Busulfan and 120 mg/kg of cyclophosphamide. The bone marrow at two weeks after transplant showed characteristic features that were unique for this clinical setting. Regenerative precursor cells with a distinct morphology commonly formed clusters or islands. Although mixed, multilinear islands were seen; the majority of islands were unilinear with a homogeneous cell content. These hematopoietic colonies were in direct proximity to degenerated cells of presumed host origin. No damage to the microenvironment was seen, the most characteristic finding being large, uniform, unilocular fat cells in close proximity to regenerative precursor cells. These morphologic changes are indicative of a complete and persistent marrow engraftment.

Microsurgical treatment of disorders of leg and foot--a report of 40 cases.

Since 1979, the authors have performed five types of microsurgical flap (including myocutaneous flap) graft to treat 40 patients with disorders of leg and foot. In the treatment of inflammatory diseases, good results were obtained owing to the following important points: 1) the operation was performed with inflammation well controlled or localized, particularly in chronic osteomyelitis; 2) indication for each type of microsurgical flap was made carefully; 3) isolation of flap was carried out gently, bleedings were thoroughly stopped by ligation.

Clinical application of the forearm island skin and fascia flap with posterior interosseous artery pedicle--report of 15 cases.

The present paper reported 15 cases, in which skin and fascia forearm island flap with posterior interosseous artery pedicle was applied. It was found that this flap has the advantages of stable vessels, abundance in blood supply and good circulation, without sacrificing the main arteries of the forearm. As a result, it is an ideal method for repair and reconstruction of the hand and forearm.