A Novel Flow Cytometric Hemozoin Detection Assay for Real-Time Sensitivity Testing of Plasmodium falciparum

Resistance of Plasmodium falciparum to almost all antimalarial drugs, including the first-line treatment with artemisinins, has been described, representing an obvious threat to malaria control. In vitro antimalarial sensitivity testing is crucial to detect and monitor drug resistance. Current assays have been successfully used to detect drug effects on parasites. However, they have some limitations, such as the use of radioactive or expensive reagents or long incubation times. Here we describe a novel assay to detect antimalarial drug effects, based on flow cytometric detection of hemozoin (Hz), which is rapid and does not require any additional reagents. Hz is an optimal parasite maturation indicator since its amount increases as the parasite matures. Due to its physical property of birefringence, Hz depolarizes light, hence it can be detected using optical methods such as flow cytometry. A common flow cytometer was adapted to detect light depolarization caused by Hz. Synchronized in vitro cultures of P. falciparum were incubated for 48 hours with several antimalarial drugs. Analysis of depolarizing events, corresponding to parasitized red blood cells containing Hz, allowed the detection of parasite maturation. Moreover, chloroquine resistance and the inhibitory effect of all antimalarial drugs tested, except for pyrimethamine, could be determined as early as 18 to 24 hours of incubation. At 24 hours incubation, 50% inhibitory concentrations (IC50) were comparable to previously reported values. These results indicate that the reagent-free, real-time Hz detection assay could become a novel assay for the detection of drug effects on Plasmodium falciparum.     

Morphological analysis of female gametophyte development in the bel1 stk shp1 shp2 mutant

Abstract

In Arabidopsis thaliana, cell fate in developing ovules is determined by the action of the homeodomain factor BELL1 (BEL1) and of the MADS-box factors SEEDSTICK (STK), SHATTERPROOF1 (SHP1) and SHP2. The analysis of the bel1 and the stk shp1 shp2 mutants revealed that the functional megaspore is formed, however, it does not proceed into megagametogenesis. In the bel1 stk shp1 shp2, quadruple mutant megasporogenesis does not take place. In this article we describe a detailed morphological analysis of the quadruple mutant, and we discuss the possibility that BELL1, STK, SHP1 and SHP2 not only control integument identity determination and development, but that they might also play a role during megasporogenesis.     

The Biopolymer Markup Language.

SUMMARY: An XML derived from a data model designed to be a hierarchical representation of an organism has been specified and a browser to use this language has been developed. AVAILABILITY: The language definition is available in HTML form at http://www.proteometrics.com/BIOML/. The BioML browser is available on request from the author.     

Patterns of Sleeping Site and Sleeping Tree Selection by Black-and-Gold Howler Monkeys (Alouatta caraya) in Northern Argentina

International Journal of Primatology - The selection of sleeping sites and sleeping trees in nonhuman primates is related to social and ecological factors. We investigate the role of body...     

Cloning of aroG, the gene coding for phospho-2-keto-3-deoxy-heptonate aldolase(phe), in Escherichia coli K-12, and subcloning of the aroG promoter and operator in a promoter-detecting plasmid

Defective transducing phages carrying aroG, the structural gene for phenylalanine (phe)-inhibitable phospho-2-keto-heptonate aldolase (EC 4.1.2.15; previously known as 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase[phe]), have been isolated, and DNA from two of these phages has been used to construct a restriction map of the region from att lambda to aroG. A 7.6-kb PstI-HindIII fragment from one of these phages was cloned into pBR322 and shown to contain aroG. The location of aroG within the 7.6 kb was established by subcloning and Tn3 transpositional mutagenesis. A fragment carrying the aroG promoter and operator has been cloned into a high copy number promoter-cloning vector (pMC489), and the resulting aroGpo-LacZ' (alpha) fusion subcloned in a low copy number vector. Strains with this fusion on the low copy number vector exhibit negative regulation of beta-galactosidase expression by both phenylalanine and tryptophan and positive regulation by tyrosine in a tyrR+ background.     

A rapid single-stranded cloning strategy for producing a sequential series of overlapping clones for use in DNA sequencing: application to sequencing the corn mitochondrial 18 S rDNA

A simple new procedure was described for producing a sequential series of overlapping clones for use in DNA sequencing. The technique used single-stranded M13 DNA and complementary DNA oligomers to form specific cleavage and ligation substrates. It was, therefore, independent of the sequence of the DNA cloned into the vector. Deletions of varying sizes were generated from one end of the insert through the 3' to 5' exonuclease activity of T4 DNA polymerase. The approximate size of the deletion and therefore the starting point for DNA sequencing could be estimated by electrophoresis of the subcloned phage DNA on a agarose gel. This greatly reduced the number of templates that must be sequenced to obtain a complete sequence. The entire procedure could be carried out in one tube in less than a day. The procedure was used to subclone and sequence the maize mitochondrial 18 S rDNA and 5' flanking region (2622 bases) in less than a week. Other applications of oligomers and single-stranded DNA in the construction of insertions, deletions, and cDNAs are discussed.     

Effect of the lymphocytosis-stimulating factor from Bordetella pertussis on murine lymphoid and hemopoietic cells

Effect of B. pertussis lymphocytosis-promoting factor (LPF) on the lympho-hematopoietic system of mice was studied. The injection of LPF was shown to sharply enhance endogenous colony formation and to induce a severe depletion of thymus cells, reaching its maximum of day 4. Thymocytes obtained on day 2 or 3 after the injection of LPF produced a suppressive effect on endogenous colony formation. The proliferative activity of hematopoietic stem cells sharply increased under the influence of LPF, though it had no radioprotective action. On the following day after the injection of LPF a steep rise in the number of hematopoietic stem cells was observed in the blood of mice: their content increased 20-fold in comparison with the control level. These data may be important for the evaluation of the side effects of pertussis vaccine on the lympho-hematopoietic system.     

The cluster method in conducting epidemiological research

The Expanded Programme on Immunization (EPI) whose goal is to reduce morbidity and mortality by providing children with immunizations against diphtheria, pertussis, tetanus, poliomyelitis, measles, and tuberculosis continually faces the problem of documenting immunization coverage rates. Therefore the EPI seeks simple, effective, and inexpensive methods of evaluation which could be implemented in different countries. An example of such a method is a simplified cluster sampling technique of estimation of immunization coverage through the examination of 210 children, selected randomly as 30 groups of 7 children each. In 1978-1984 more than 1000 immunization coverage surveys were performed all over the world, mainly in developing countries. In a modified way this method is also used to collect data on morbidity and mortality of certain EPI target diseases as well as diarrhoeal diseases.