Molecular Mechanisms of Translation Initiation in Mammals Studied by in vitroReconstruction of Initiation Complexes

Papers on the mechanisms of translation initiation in mammals studied by reconstruction of initiation complexes from individual components are reviewed. The author points to the constraints of this approach and to the pitfalls ignoring which one might come to erroneous conclusions and even artifacts. In addition, some methods employed in the field as well as some technical problems are discussed in the paper, together with the means of obviating them. The review could be a guidebook for newcomers into this quite labor-consuming field.     

Evidence that free GPI glycolipids are essential for growth of Leishmania mexicana.

The cell surface of the parasitic protozoan Leishmania mexicana is coated by glycosylphosphatidylinositol (GPI)-anchored glycoproteins, a GPI-anchored lipophosphoglycan and a class of free GPI glycolipids. To investigate whether the anchor or free GPIs are required for parasite growth we cloned the L.mexicana gene for dolichol-phosphate-mannose synthase (DPMS) and attempted to create DPMS knockout mutants by targeted gene deletion. DPMS catalyzes the formation of dolichol-phosphate mannose, the sugar donor for all mannose additions in the biosynthesis of both the anchor and free GPIs, except for a alpha1-3-linked mannose residue that is added exclusively to the free GPIs and lipophosphoglycan anchor precursors. The requirement for dolichol-phosphate-mannose in other glycosylation pathways in L.mexicana is minimal. Deletion of both alleles of the DPMS gene (lmdpms) consistently resulted in amplification of the lmdpms chromosomal locus unless the promastigotes were first transfected with an episomal copy of lmdpms, indicating that lmdpms, and possibly GPI biosynthesis, is essential for parasite growth. As evidence presented in this and previous studies indicates that neither GPI-anchored glycoproteins nor lipophosphoglycan are required for growth of cultured parasites, it is possible that the abundant and functionally uncharacterized free GPIs are essential membrane components.     

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.     

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.     

Predicted class-I aminoacyl tRNA synthetase-like proteins in non-ribosomal peptide synthesis

Background  

Recent studies point to a great diversity of non-ribosomal peptide synthesis systems with major roles in amino acid and co-factor biosynthesis, secondary metabolism, and post-translational modifications of proteins by peptide tags. The least studied of these systems are those utilizing tRNAs or aminoacyl-tRNA synthetases (AAtRS) in non-ribosomal peptide ligation.     

Action of pilocarpine on the normal frog heart and in pathology

Experiments on frogs were performed to examine the effect of the M-cholinomimetic pilocarpine on the heart. It was discovered that at concentrations of 10(-15)--10(-5) g/ml pilocarpine exerted only an adverse chronotropic effect on the perfused heart. When applied at a concentration of 10(-4) g/ml the drug produced a negative as well as a positive chronotropic effect. The latter occurred spasmodically (without progressive rise in the heart rate) in association with a slow heart rate. In some experiments such effects were preceded by a certain deceleration of the heart. In experiments with positive chronotropic effects, arrhythmias and sinoatrial dissociation were observed sometimes. Experiments with recording of the electrograms of the sinuses and lower parts showed that such effects were caused not by pacemaker acceleration but by the removal of the blockade of conduction, between the pacemaker and the atria. As far as the pacemaker is concerned, pilocarpine exerted only a negative chronotropic effect.     

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.