Update on the concept of the cell cycle: the contribution of flow cytometry

Flow cytometry has been extensively used to provide accurate estimates of the relative amounts of various cellular constituents (DNA, RNA, proteins) for cell kinetic studies. Multiparametric analysis also supports the recent concept that cell growth and the DNA division cycle may be under distinct regulatory mechanisms. Moreover, metabolic subcompartments of the cell cycle, distinguished by flow cytometry, have offered a highly sensitive cell classification in comparison with the conventional distinction of the four main phases of the cell cycle. Finally, a new sensitive and powerful technology, BrdU/DNA analysis, represents a remarkable maturing of a very useful alternative for the study of DNA synthesis and cell cycle traverse.     

The contribution of Plasmodium chabaudi to our understanding of malaria

Malaria kills close to a million people every year, mostly children under the age of five. In the drive towards the development of an effective vaccine and new chemotherapeutic targets for malaria, field-based studies on human malaria infection and laboratory-based studies using animal models of malaria offer complementary opportunities to further our understanding of the mechanisms behind malaria infection and pathology. We outline here the parallels between the Plasmodium chabaudi mouse model of malaria and human malaria. We will highlight the contribution of P. chabaudi to our understanding of malaria in particular, how the immune response in malaria infection is initiated and regulated, its role in pathology, and how immunological memory is maintained. We will also discuss areas where new tools have opened up potential areas of exploration using this invaluable model system.     

The contribution of cell cycle regulation to endosperm development

Development of the seed endosperm involves several different types of coordinated cell cycle programs: acytokinetic mitosis, which produces a syncytium soon after fertilization; cellularization through the formation of modified phragmoplasts; cell proliferation, in which mitosis is coupled to cell division; and, in certain species like cereal crops, endoreduplication. Understanding the regulation of these programs and their transitions is challenging, but it has the potential to define important links between the cell cycle, cell differentiation and development, as well as provide tools for the manipulation of seed yield. A relatively large number of mutants display endosperm proliferation defects, and connections with known cell cycle genes are beginning to emerge. For example, it is becoming increasingly evident that the master cell cycle regulators, the cyclin-dependent kinases and retinoblastoma-related families, play key roles in the events leading to endosperm formation and development. Recent studies highlight cross-talk between pathways controlling the cell cycle and genomic imprinting.     

The potential contribution of mass treatment to the control of Plasmodium falciparum malaria

Mass treatment as a means to reducing P. falciparum malaria transmission was used during the first global malaria eradication campaign and is increasingly being considered for current control programmes. We used a previously developed mathematical transmission model to explore both the short and long-term impact of possible mass treatment strategies in different scenarios of endemic transmission. Mass treatment is predicted to provide a longer-term benefit in areas with lower malaria transmission, with reduced transmission levels for at least 2 years after mass treatment is ended in a scenario where the baseline slide-prevalence is 5%, compared to less than one year in a scenario with baseline slide-prevalence at 50%. However, repeated annual mass treatment at 80% coverage could achieve around 25% reduction in infectious bites in moderate-to-high transmission settings if sustained. Using vector control could reduce transmission to levels at which mass treatment has a longer-term impact. In a limited number of settings (which have isolated transmission in small populations of 1000-10,000 with low-to-medium levels of baseline transmission) we find that five closely spaced rounds of mass treatment combined with vector control could make at least temporary elimination a feasible goal. We also estimate the effects of using gametocytocidal treatments such as primaquine and of restricting treatment to parasite-positive individuals. In conclusion, mass treatment needs to be repeated or combined with other interventions for long-term impact in many endemic settings. The benefits of mass treatment need to be carefully weighed against the risks of increasing drug selection pressure.     

Innate resistance to malaria: the intraerythrocytic cycle

The human innate resistance to P. falciparum malaria is based on genetic features that affect several stages of the intraerythrocytic cycle of the plasmodia. HbS, HbE and alpha and beta thalassemia (in addition to G-6PD deficiency) are protective to the carriers, because they inhibit the intraerythrocytic growth period, and in the case of AS red cells, in addition, parasitosis make them detectable expeditiously by the spleen. Blood group polymorphisms can interfere with red cell invasion by plasmodia. HbC belongs to a special category, since it apparently interferes with the cycle at the moment of cell lysis and release of merozoites. Finally, ovalocytosis observed in South East Asia, which most likely corresponds to a cytoskeleton or membrane protein defect, protects from malaria by inhibiting invasion. It should be kept in mind that many of these red cell defects might protect individuals in the critical first 5 years of life by retarding the switch of HbF to adult hemoglobin, since the HbF containing red cells are less than hospitable to the parasite.     

The contribution of residues 192 and 193 to the specificity of snake venom serine proteinases

Snake venom serine proteinases, which belong to the subfamily of trypsin-like serine proteinases, exhibit a high degree of sequence identity (60-66%). Their stringent macromolecular substrate specificity contrasts with that of the less specific enzyme trypsin. One of them, the plasminogen activator from Trimeresurus stejnegeri venom (TSV-PA), which shares 63% sequence identity with batroxobin, a fibrinogen clotting enzyme from Bothrops atrox venom, specifically activates plasminogen to plasmin like tissue-type plasminogen activator (t-PA), even though it exhibits only 23% sequence identity with t-PA. This study shows that TSV-PA, t-PA, and batroxobin are quite different in their specificity toward small chromogenic substrates, TSV-PA being less selective than t-PA, and batroxobin not being efficient at all. The specificity of TSV-PA, with respect to t-PA and batroxobin, was investigated further by site-directed mutagenesis in the 189-195 segment, which forms the basement of the S(1) pocket of TSV-PA and presents a His at position 192 and a unique Phe at position 193. This study demonstrates that Phe(193) plays a more significant role than His(192) in determining substrate specificity and inhibition resistance. Interestingly, the TSV-PA variant F193G possesses a 8-9-fold increased activity for plasminogen and becomes sensitive to bovine pancreatic trypsin inhibitor.     

Spatial simulation of malaria transmission and its control by malaria transmission blocking vaccination

A simple, visual representation of spatial aspects of malaria transmission in successive snap-shots in time, is presented. The spatial components of the simulation involve (i) the identification of mosquito vector breeding sites of defined shape and area, (ii) the identification of a zone of malaria transmission determined by the shapes and areas of the vector breeding sites and the distance from these sites that the mosquitoes disperse, (iii) a human population dispersed in relation to the malaria transmission zone, (iv) perimeters around each individual human within which his or her infection can be transmitted by the local vector mosquitoes. The intensity of transmission within a malaria transmission zone is given by a number which is the number of new cases of malaria that each existing case will distribute through the human population within the duration of an infection. The simulation has been used here to examine the effects of vaccination against malaria transmission. Different levels of vaccine coverage are represented under endemic and epidemic malaria. The consequences of full or partial coverage of a zone of malaria transmission are also examined. The results are numerically compatible with the predictions of previous simple mathematical simulations of malaria transmission and interventions. The present simulation allows the nature of malaria transmission and the effects of interventions to be communicated easily and directly to an audience. It could have practical value in discussions of malaria control strategies with health planners.     

Ultramicrobacteria: Formation of the concept and contribution of ultramicrobacteria to biology

Ultramicrobacteria (UMB) are species of the domain Bacteria characterized by very small sizes of proliferating cells (less than 0.1 μm³ in volume) and small genomes (3.2 to 0.58 Mb). Some authors use the term nanobacteria as a synonym of UMB. Several tens of UMB species have been isolated from various natural habitats: sea water, soil, silt, Greenland ice sheet, permafrost soils, and intestines of humans and insects. Under laboratory conditions, they are cultivated on different nutrient media. In the second prokaryotic domain, the Archaea, ultrasmall forms (ultramicroarchaea) have also been described, including nanoarchaea (members of the genus Nanoarchaeum) with a cell volume of less than 0.1 μm³. The term nanobacteria is used in the literature also to denote ultrasmall bacterium-like particles occurring in rocks, sands, soils, deep sub-surface layers, meteorites, and clinical samples. The systematic position and the capacity for self-reproduction of these particles are still unclear. The cultured UMB forms are characterized by highly diverse morphology, ultrastructural organization, physiology, biochemistry, and ecology. UMB form three groups according to the type of cell wall structure and the reaction to Gram staining: (1) gram-negative, (2) gram-positive, and (3) cell wall-lacking. Their cells divide by constriction, septation, or budding. The unique processes performed by UMB are dehalorespiration and obligate or facultative epibiotic parasitism. The UMB that synthesize organic compounds in ocean waters with the involvement of proteorhodopsin play a great role in the biosphere. UMB have been found in seven large phylogenetic groups of prokaryotes, where their closest relatives are organisms with larger cells typical of bacteria, which is evidence of the polyphyletic origin of the currently known UMB species and the reductive mode of their evolution.     

The contribution of plant biology to the concept of virus (1886-1917).

Between 1860 and 1880 the so-called "theory of the infective germ", which stated in final way that every infectious disease was produced by a living pathogen agent, achieved great consent. The criteria of determining the presence of infectious pathogens (fungi, bacteria, protozoa) were established by "Koch's postulate", a set of experimental procedures conceived for isolating and determining single pathogens. In the last quarter of the 19th century became however evident that the agents of severe infectious diseases could not be identified through the "postulates". These agents could not be seen in light microscopy nor cultured in vitro but could pass through the thin pores of filters which hold back cellular micro-organisms. This last characteristic became a selective method to recognise these peculiar agents, from then named "filterable viruses". Most microbiologists considered the filterable viruses as living micro-organisms because of their extraordinary capacity of in vivo proliferation, and the impossibility of pointing out their structures was due to limits of the experimental techniques. Between the end of the 19th century and 1917, four plant biologists suggested that the filterable viruses were complex chemical substances rather than cellular microorganisms. Their contribution, not appreciated by the contemporary colleagues, laid the foundation of the modern concept of virus.     

Active site contribution to specificity of the aspartic proteases plasmepsins I and II

Plasmepsins I and II (PM I and II) are aspartic proteases involved in the initial steps of Plasmodium hemoglobin degradation. They are attractive targets for antimalarial drug development. The two enzymes are 73% identical, yet have different substrate and inhibitor specificities. The x-ray structures of proform and mature PM II have been determined, but models of PM I do not adequately explain the selectivity of the two proteases. To better understand the basis of these recognition differences, we have identified nine residues of PM II that are in proximity to the inhibitor pepstatin in the crystal structure and differ in PM I. We mutated these residues in PM II to the cognate amino acids of PM I. Kinetic parameters for substrate and inhibitors for the PM II-mutant were similar to those of PM II-wild type (WT). Cleavage specificity was assessed using hemoglobin or a random decamer peptide library as substrate. Again, PM II-mutant behaved like PM II-WT rather than PM I-WT. These results indicate that differences in plasmepsin specificity depend more on conformational differences from distant sites than on specific active site variation.     

Quantitating tissue specificity of human genes to facilitate biomarker discovery

We describe a method to identify candidate cancer biomarkers by analyzing numeric approximations of tissue specificity of human genes. These approximations were calculated by analyzing predicted tissue expression distributions of genes derived from mapping expressed sequence tags (ESTs) to the human genome sequence using a binary indexing algorithm. Tissue-specificity values facilitated high-throughput analysis of the human genes and enabled the identification of genes highly specific to different tissues. Tissue expression distributions for several genes were compared to estimates obtained from other public gene expression datasets and experimentally validated using quantitative RT-PCR on RNA isolated from several human tissues. Our results demonstrate that most human genes ( approximately 98%) are expressed in many tissues (low specificity), and only a small number of genes possess very specific tissue expression profiles. These genes comprise a rich dataset from which novel therapeutic targets and novel diagnostic serum biomarkers may be selected.