PubMed · 5540190
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D R Seaton. 1971. Amoebicides.. https://pubmed.ncbi.nlm.nih.gov/5540190/
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Manipulation of gene expression in Giardia lamblia, one of the most ancient eukaryotes, may provide insights into the evolutionary transition from prokaryotes to eukaryotes. Two recent successes in transient expression of the firefly luciferase (luc) gene in G. lamblia were mediated by a 5'-untranslated region (UTR) of the Giardia glutamate dehydrogenase (gdh) gene and a giardiavirus (GLV) genomic transcript, respectively. We now report a stable coexpression of luc gene with a neomycin phosphotransferase (neo(r)) gene in G. lamblia. An in vitro transcript of the construct pC670-Neo; containing the neo(r) encoding region flanked with the 5'670 nucleotides (nt) and the 3'2022 nt portion of GLV positive strand RNA, was electroporated into G. lamblia trophozoites that were infected with GLV. G418-resistant Giardia trophozoites were cloned, and the neo(r) mRNA in these clones was found to increase with increasing G418 pressure. This drug resistance remained stable upon continuous in vitro cultivation in the absence of G418 for over 15 days. Another plasmid pNeo/GDH/Luc, was constructed by inserting luc gene downstream from the neo(r) gene and the 193 nt 5' portion of gdh gene in pC670-Neo, and its bicistronic in vitro transcript was introduced into GLV-infected G. lamblia by electroporation. The transfectants demonstrated G418-resistance and persistent luciferase activity at levels parallel to the amount of G418 used for selection, peaking at a level of several thousand-fold above the background. Taken together, these data indicate that the neo(r) gene provides an effective selection marker for transformation of Giardia trophozoites, and the bicistronic RNA transfection vector may open the way for functional analysis of other genes in Giardia.
It has been shown that oxidation of polyamines by polyamine oxidases can produce toxic compounds (H2O2, aldehydes, ammonia) and that the polyamine oxidase-polyamine system is implicated, in vitro, in the death of several parasites. Using Amoeba proteus as an in vitro model, we studied the cytotoxicity to these cells of spermine, spermidine, their acetyl derivatives, and their hypothetical precursors. Spermine and N1-acetylspermine were more toxic than emetine, an amoebicidal reference drug. Spermine presented a short-term toxicity, but a 48-h contact time was necessary for the high toxicity of spermidine. The uptake by Amoeba cells of the different polyamines tested was demonstrated. On the other hand, a high polyamine oxidase activity was identified in Amoeba proteus crude extract. Spermine (theoretical 100%) and N1-acetylspermine (64%) were the best substrates at pH 9.5, while spermidine, its acetyl derivatives, and putrescine were very poorly oxidized by this enzyme (3-20%). Spermine oxidase activity was inhibited by phenylhydrazine (nil) and isoniazid (approximately 50%). Mepacrine did not inhibit the enzyme activity at pH 8. Neither monoamine nor diamine oxidase activity (approximately 10%) was found. It must be emphasized that spermine, the best enzyme substrate, is the most toxic polyamine. This finding suggests that knowledge of polyamine oxidase specificity can be used to modulate the cytotoxicity of polyamine derivatives. Amoeba proteus was revealed as a simple model for investigation of the connection between cytotoxicity and enzyme activity.
Observations were made of 996 encounters between children with diarrhoea and practitioners (28 paediatricians, 62 general practitioners) in Karachi, Pakistan. Oral rehydration salt (ORS) was prescribed in more than 50% of encounters by 53% of general practitioners (GPs) and 61% of paediatricians. Sixty-six percent of GPs and 50% of paediatricians prescribed antibacterials, 60% of GPs and 28% of paediatricians prescribed antidiarrhoeals and 39% of GPs and 32% of paediatricians prescribed antiamoebics in more than 30% of their encounters. Looking at all the encounters, we observed that ORS was prescribed in 52 and 51%, antibacterials in 41 and 36%, antidiarrhoeals in 48 and 29%, and antiamoebics in 26 and 22% of encounters by GPs and paediatricians, respectively, Cotrimoxazole was the most frequently prescribed antibacterial by both types of practitioners. Antidiarrhoeals were prescribed more often by GPs than by paediatricians. In 77% of their encounters, GPs dispensed drug formulations known as "mixtures' made in their own dispensing corners. The mean duration of encounters between patients and GPs was 3 +/- 2 minutes and between patients and paediatricians was 9 +/- 4 minutes. These results indicate inadequate prescription of ORS and excessive prescription of antibacterials, antidiarrhoeals and antiamoebics. Intervention strategies need to be planned to improve the prescribing practices of both groups.