ABSTRACT
To insight into genetic differences between chloroquine-resistant line (RC) and chloroquine-sensitive strain (N) of Plasmodium berghei. MethodsAfter continous cbloroquine-pressure upon RC line at higher dosage (50mg/kg. d) ,total RNAs from the RC line and the N strain of P. berghei were isolated for simplified differential display reverse transcript polymerase chain reaction (sDDRT-PCR ). The generated differential fragments had been repetitively rescued and identified by PCRs before one pair of suspected differential fragments (N25 and R25 )were cloned and sequenced. Then, their sequences were analyzed through PC-gene program and BLAST search. ResultsThough the identity of two nucleotide sequences of N252 and R251 ,cloned separately from the N25 and R25 fragments, were up to 99.8％ ,their predicted amino acid secondary structures were quite different due to multiple mutations. Compared with the published sequences in GeneBank,EMBL,DDBJ and PDB database ,no similar gene was found ,using BLAST search. However their partial nucleotide sequences (62nt from query 128nt to189nt bore highly homology to part sequence(from 1053nt to1114nt)of rattus norvegicus mRNA for phospholipase B,up to 93.5％ in N251 and 91.9％ in N252 respectively. ConclusionIt is feasible to isolate chloroquine-resistant related genes by using simplified DDRT-PCR combined repetitively rescuing and PCR identifying the interest differential DNAs together with sequence analyses.
ABSTRACT
Objective: To study the mechanism that erythrocytic stages Plasmodium berghei chloroquine resistant strain (RC strain) were eliminated in their hosts. Methods: Interactions of leukocytes with the parasites in mice livers infected with the RC strain or chloroquine sensitive strain (N strain) of P. berghei were studied by electron transmission microscopy. Results: None of leukocytes proliferated and infiltrated in mice livers infected with the N strain. Whereas in mice livers infected with the RC strain, proliferation and infiltration of monocyte macrophages, lymphocytes and nuetrophils occurred within portal areas and hepatic sinusoids with sequestration of numerous parasitized erythrocytes. The activated monocyte macrophages adhered to the parasitized red blood cells with their surface membranes. A lot of the parasites became crisis form within the parasitized erythrocytes which directly contacted with the phagocytes or not. Phagocytosis of free merozoites by macrophages was rarely revealed, but of the whole parasitized erythrocytes was not found. Conclusion: The crisis form of the parasites induced by the activated monocyte macrophages, rather than the direct phagocytolysis, is mainly responsible for the elimination of the P. berghei RC strain in their hosts. [