[Study on molecular phylogeny of Schistosoma sinensium based on nuclear ribosomal DNA].

OBJECTIVE: To determine the phylogenetic relationships between Schistosoma sinensium and other Schistosomatid species using DNA sequence data. Two segments of the nuclear rDNA repeat, the second internal spacer (ITS2) and large subunit (LSU/12S) were selected for sequencing. METHODS: Adult worms stored in 100% methanol were washed 3 times with 0.1 x TE (pH8.0) and the genomic DNA was extracted by the GNT-K method. The target regions were amplified by PCR using specific primers. The PCR products were purified before ligation into the plasmid pT-adv (Clontech). Recombinant plasmids were amplified in E. coli (strain TOP10), extracted and purified using routine methods and then sequenced using M13 primers (F/R) on a Licor long-read auto-sequencer. Sequences of related schistosomes were retrieved from GenBank and aligned with our data in the sequence editor ESEE. Gene trees were constructed in PHYLIP (Version 3.6 alpha July, 2,000) and MEGA (version 2.0 beta build 3) using both Maximum Parsimony and Neighbor-Joining methods. For parsimony analysis, all characters were treated as unordered and with equal weights. At least 3,000 cycles of bootstrapping were carried out. For analysis in MEGA, all gap columns were deleted. Schistosomatium douthitti and Trichobilharzia were used as outgroups. RESULTS: The ITS2 and LSU sequences of Schistosoma sinensium were obtained. The ITS2 sequence of Trichobilharzia sp. was reported here for the first time. CONCLUSION: The phylogenetic trees from these data of nuclear rDNA suggested that S. sinensium belongs to the Asian schistosome group. And this species might be an ancient member in the Asian clade.

[Preliminary study on cytochrome C oxidase 1 gene of Oncomelania hupensis from Miao River area in Hubei province].

OBJECTIVE: To study the mitochondrial cytochrome C oxidase 1(CO1) gene of Oncomelania snails from Miao River area in Hubei Province. METHODS: Oncomelania snails were collected from Miao River area, including upstream and downstream. Genomic DNA was extracted from the tissue of the snail. PCR was used to amplify a fragment of the CO1 gene. Sequences of the CO1 fragment were determined directly from the purified PCR products by an automated sequencer. Sequences for each individual were assembled and edited using ESEE 3.0 s. A distance matrix was computed using program DNADISt of PHYLIP(3.57). Unrooted maximum likelihood trees were calculated from program FITCH. RESULTS: The amplified CO1 gene of the snail was a fragment of 638 bp in length. Sequence analysis showed that the accumulated variable sites were significant different between upstream and downstream populations, being 29 and 46, respectively. From the number of variable sites in the gene, snails in this area were roughly separated into two groups. Each of them was a mixture of both upstream and downstream snails. Same haplotypes were confirmed to be present among the collected sites along the river. From the distance matrix of sequence divergence, the population upstream vs downstream differed by 0.0221 +/- 0.0105. CONCLUSION: There were more variation in downstream population than that in upstream. Gene flow was identified in these populations. The phylogenetic trees suggest the existence of two groups, but all of them belong to 0. h. hupensis.

[The Inhibitory Study of TNP 470 Combined CDDP on the Tumor Growth and Lung Metastasis of Adenoid Cystic Carcinoma ACC M in Nude Mice]

OBJECTIVE:To study the effect of TNP-470 combined CDDP on the tumor growth and lung metastasis of cell strain ACC-M in vivo. METHODS:High potential lung metastatic adenoid cystic carcinoma cell strains (ACC-M) 5x10(5) were injected simultaneously to 18 nude mice subcutaneously and enously. The animals were randomly divided into 3 groups. In group 1, TNP-470 was administered subcutaneously at the dose of 30mg/kg every other day for 2 weeks from the beginning of 3(rd) week to the end of 4(th) week. In group 2, 5mg/kg CDDP were injected into peritoneal cavity at the beginning of 3(rd) week and 4(th) week and TNP-470 in the same manner as group 1 additionally. In group 3, the same volume of 0.97% NaCl was injected in the manner of group 1. The mice were sacrificed by dislocating the neck at the end of 4 weeks. The tumor size and weights were recorded. The vascular density was measured by rabbit-anti-human factor VIII antibody stain. The results were analyzed by Student t and X(2) test. RESULTS:Group 1 and group 2 had fewer tumor weight and lung metastasis than the 3(rd) group. There was significant difference in the tumor weight and lung metastasis between group 1 and group 2. But the vascular density had no difference among three groups. CONCLUSION:TNP-470 combined CDDP has stronger inhibition on both the tumor growth and lung metastasis of human adenoid cystic carcinoma ACC-M than used alone.

Genetic differentiation among three populations of Anopheles minimus of Guangxi and Yunnan Provinces in the People's Republic of China.

Electrophoretic studies were carried out on isozymes of 3 populations of Anopheles minimus collected from Guangxi and Yunnan Provinces of the People's Republic of China in 1993. Eight proteins were analyzed by 5% polyacrylamide gel electrophoresis. The most variable population, Y-F, was highly polymorphic at 14 of 20 loci (P=0.700) with an average heterozygosity H of 0.340. P values of 0.500 and 0.700, and H values of 0.220 and 0.210 were obtained for each from 'Guangxi-Lab' (GX-L) and 'Yunnan-Lab' (Y-L), respectively. Nei's genetic distances (D) between Y-L and GX-L, Y-F and GX-L, and Y-F and Y-L were 0.1131, 0.1946 and 0.1069, respectively. These results suggest that GX-L is distant from the 2 other populations, Y-L and Y-F, and that this genetic differentiation between the 2 populations of Yunnan and Guangxi Provinces corresponds to the forms A and B, which were morphologically classified by Xu et al (unpublished).

[Effect of spermidine on uptake of chloroquine by Plasmodium berghei].

To probe into the effect of spermidine on chloroquine (Chl) uptake by P berghei and its role of Chl-resistance, mice infected with Chl sensitive strain (CS) of P berghei were given Chl 20 mg.kg-1 ig combined with spermidine (Spe) 42 mg.kg-1 ip. It was found that 3 and 16 h after combined administration, Chl quantity uptaken by the parasites was reduced respectively by 59.6% and 53.8% in comparison with that in the Chl group. However, there was no difference in parasitaemia between Chl group (2.3 +/- 1.0) and Chl-Spe group (1.7 +/- 1.0), whereas the untreated control group remained a parasitaemia of 36 +/- 9. The authors deemed that Chl resistance is not merely attributed to the insufficient quantity of Chl in the Chl resistant parasites, the change in the sensitivity to Chl of Chl resistant parasites and the role of Spe in Chl resistance production should also be taken into consideration.

[Alteration in resistance of Plasmodium falciparum to chloroquine after cessation of chloroquine medication for twelve years].

In view of the fact the resistance of Plasmodium falciparum to chloroquine occurred extensively in Hainan, a decision was made in 1979 that the use of chloroquine should be quit in the whole province. A longitudinal survey on chloroquine-sensitivity of P. falciparum was carried out during 1981-1991 to observe the variation in resistance of the parasite after the cessation of the chloroquine medication for every 2-3 years. A tendency of progressive decline of resistance was revealed. By using in vitro test, the rate of chloroquine-resistant P. falciparum dropped from 97.9% in 1981 to 60.9% in 1991 (P < 0.001). The mean dosage of chloroquine for complete inhibition of schizont formation declined from 10.46 pmol/microliters in 1981 to 3.02 pmol/microliters in 1991 (P < 0.001). The percentage of population requiring larger dosage (6.4 pmol/microliters to completely inhibit schizont formation declined from 83.3% in 1981 to 17.4% in 1991 (P < 0.001); whereas those requiring small dosage (1.6 pmol/microliters), increased from 4.2% in 1981 to 60.8% in 1991 (P < 0.001). In in vivo test, the rate of chloroquine-resistant P. falciparum decreased from 84.2% in 1981 to 40% in 1991 (P < 0.001). The proportion of RII plus RIII cases of the total resistant cases dropped from 59.4% in 1981 to 37.5% in 1991 (0.02 > P > 0.01).

[Preliminary study on the surface antigen gene of Plasmodium malariae].

The genomic Hind III lambda gt1149 library from Plasmodium malariae was screened with two types of oligonucleotides, both of which were from the P190 gene of P. falciparum. One, MAD20 type, was made from spanning nucleotides 4488-4562 in the P190 gene of MAD20 strain and another, K1 type, was made from spanning nucleotides 3652-3692 in the P190 of K1 strain. Both were end-labelled with 32P-ATP as probes before hybridization. Two clones were selected. One clone, designated lambda MSA-1, was specifically recognized by the MAD20 type oligo probe; the other, designated lambda MSA-2, by the K1 type oligo probe. lambda MSA-1 and lambda MSA-2 inserts were obtained by Hind III digestion of the two clone phage DNA. The analysis of the two inserts showed that the size of lambda MSA-1 is 5.5 kb whilst that of lambda MSA-2 is 3.5kb. The MSA-1 and MSA-2 inserts recloned into PUC8 were digested with the restriction enzymes Bg1 II, EcoR I, Xba I, Hind II and Pst I. The results showed that the MSA-1 DNA had one Bg1 II site, one EcoR I site, two Xba I sites, one Hind II site and one Pst I site. The MSA-2 DNA had only one Hind II site. The surface antigen gene of P. malariae was little known. This result also showed that there was probably an analogue of P190 on the surface of P. malariae, and they might fall into two types. This study is informative for further investigation on malariae parasites.

[A handy medium for in vitro assessment of sensitivity of Plasmodium falciparum to chloroquine and mefloquine in the field].

The use of liquid RPMI 1640 medium (added NaHCO3 and serum) in sealed ampoule for in vitro assessment of sensitivity of Plasmodium falciparum to chloroquine and meflopuine in the field was very successful. On chloroquine plates with WHO supplied medium, 9 of 13 isolates were interpretable (successful rate 69.2%), the maturation rate from ring-forms to schizonts was 53.3%, the average number of nuclei per schizont 5.1. With lyophilized medium the successful rate was 100%, the maturation rate 65.9%, the average number of nuclei per schizont 7.4, while with medium in sealed ampoule, the corresponding figures were 92.3%, 65.1% and 7.3 respectively. On mefloquine plates with WHO supplied medium, 7 of 11 isolates were interpretable (63.6%). In control wells, 52.3% of schizonts matured, the number of nuclei per schizont was 5.1. With lyophilized medium the successful rate was 100%, the maturation rate 61.9%, the number of nuclei was 8.1, while those with liquid medium in sealed ampoule were 100%, 59.8% and 7.5 respectively. The results showed that the liquid medium in sealed ampoules stored within 56 days at 4 degrees C could still support the growth of Plasmodium falciparum, its supporting effect being better than that of WHO standard medium, but similar to lyophilized medium. The liquid medium in sealed ampoule had the advantages of easy carrying time-saving and more applicability under field conditions.