Long-term in vitro cultures of Plasmodium berghei and preliminary observations on gametocytogenesis

Several long-term in vitro cultures of the rodent malaria parasite Plasmodium berghei were established. In these cultures, ranging over 17–90 days, peak parasitaemias of over 20% and multiplication rates of up to 7·7 were observed. A previously described culture method was used. The method for medium refreshment was changed and rat erythrocytes were used as host cells. The long-term cultivation of Plasmodium berghei enables us to study the process of gametocytogenesis since male and female gametocytes were produced in all cultures and reached full maturity, demonstrated by exflagellation and fertilization in vitro.     

Ancient host-pathogen associations maintained by specificity of chemotaxis and antibiosis

Switching by parasites to novel hosts has profound effects on ecological and evolutionary disease dynamics. Switching requires that parasites are able to establish contact with novel hosts and to overcome host defenses. For most host–parasite associations, it is unclear as to what specific mechanisms prevent infection of novel hosts. Here, we show that parasitic fungal species in the genus Escovopsis, which attack and consume the fungi cultivated by fungus-growing ants, are attracted to their hosts via chemotaxis. This response is host-specific: Escovopsis spp. grow towards their natural host cultivars more rapidly than towards other closely related fungi. Moreover, the cultivated fungi secrete compounds that can suppress Escovopsis growth. These antibiotic defenses are likewise specific: in most interactions, cultivars can inhibit growth of Escovopsis spp. not known to infect them in nature but cannot inhibit isolates of their naturally infecting pathogens . Cases in which cultivars are susceptible to novel Escovopsis are limited to a narrow set of host–parasite strain combinations. Targeted chemotactic and antibiotic responses therefore explain why Escovopsis pathogens do not readily switch to novel hosts, consequently constraining long-term dynamics of host–parasite coevolution within this ancient association.     

Commitment to gametocytogenesis in Plasmodium falciparum

To achieve transmission, a subpopulation of asexually dividing bloodstream forms of the human malaria parasite Plasmodium falciparum withdraws from the cell cycle to develop into gametocytes - cells specialized for sexual reproduction and invasion of the mosquito vector. For natural selection to maximize transmission to new hosts, a balance must have evolved between asexual replication and sexual differentiation. Here, Mike Dyer and Karen Day consider observations on the process of commitment to gametocytogenesis and use this information as the framework for a model that begins to explain the control of the dynamics between asexual and sexual development.     

Apicoplast and mitochondrion in gametocytogenesis of Plasmodium falciparum

Live cell imaging of human malaria parasites Plasmodium falciparum during gametocytogenesis revealed that the apicoplast does not grow, whereas the mitochondrion undergoes remarkable morphological development. A close connection of the two organelles is consistently maintained. The apicoplast and mitochondrion are not components of the male gametes, suggesting maternal inheritance.     

The reaggregation of adult rat liver cells maintained in vitro

The reaggregation of dissociated adult rat liver cells maintained in vitro for up to 96 h is described. Cultures were examined by dark-ground fluorescence and electron microscopy. Spaces resembling bile canaliculi were formed between reaggregated hepatocytes. Desmosomes and ‘tight’ junctions were formed between hepatocytes but ‘gap’ junctions were not detected. In older cultures structural damage was observed in many hepatocytes and some of them ingested cell debris by phagocytosis. Structures resembling bile ducts and sinusoids were also formed but complex association between all three types of cell aggregate was not observed.     

Problems with continuous-time malaria models in describing gametocytogenesis

Most mathematical models of malaria infection represent parasites as replicating continuously at a constant rate whereas in reality, malaria parasites replicate at a fixed age. The behaviour of continuous-time models when gametocytogenesis is included, in comparison to a more realistic discrete-time model that incorporates a fixed replication age was evaluated. Both the infection dynamics under gametocytogenesis and implications for predicting the amount parasites should invest into gametocytes (level of investment favoured by natural selection) are considered. It is shown that the many malaria models with constant replication rates can be represented by just 3 basic types. For these 3 types, it is then shown that under gametocytogenesis (i) in 2 cases, parasite multiplication and gametocyte production is mostly much too low, (ii) in the third, parasite multiplication and gametocyte production is mostly much too high, (iii) the effect of gametocyte investment on parasite multiplication is mostly too high, (iv) the effect of gametocyte investment on gametocyte production is nearly always too low and (v) with a simple approximation of fitness, the predicted level of gametocyte investment is mostly much too low. However, a continuous model with 48 age-compartments compares well to the discrete model. These findings are a further argument for modelling malaria infections in discrete time.     

Schistosoma japonicum: changes in lipase activity in adults maintained in vitro

Schistosoma japonicum (Chinese strain) adult worm pairs (10-12 weeks old) were fixed in 4 degrees C absolute acetone or neutral buffered formalin either immediately after recovery from mice, or following incubation for 0.5 or 1.0 h. Males and females were incubated individually in Earle's balanced salt solution at 37 degrees C. Lipase activity was determined in frozen sections by Gomori's method using Tween 80 as substrate. In unincubated males, lipase activity was localized only in the parenchyma; in females it was present in the vitellaria and parenchyma subjacent to the tegument. After 0.5 h incubation, males showed less lipase activity in the parenchyma than unincubated males, and females showed increased activity in the parenchyma subjacent to the tegument, but not in the vitellaria. Following 1.0 h incubation, males showed less lipase activity than previously, and females showed a marked increase in activity, particularly in the parenchyma subjacent to the tegument.     

Survival characteristics of normal differentiated rat thyroid cells maintained in vitro

The radiation response of a cultured line of differentiated rat thyroid epithelial cells (FRTL-5) was determined by clonogenic assay in vitro and compared to the previously reported response curve for primary rat thyroid cells assayed by transplantation in vivo. The beta components (or D0 estimates) for the two sets of data were not significantly different although the response of the FRTL-5 cells was displaced to the right as a consequence of differences in the shoulder of the survival curves. These results indicate that this in vitro model can be used to study the radiation response of thyroid epithelial cells and by extrapolation perhaps other hormonally responsive, slowly proliferative parenchyma. The cell age response for FRTL-5 cells was determined after separation of the cells into various phases of the cell cycle by centrifugal elutriation. Resistant peaks in mid-G1 and G2M phases of the cell cycle were detected. The cell age response data for other portions of the cell cycle should be interpreted cautiously because less than satisfactory enrichments were achieved.     

Functioning of plant-bacterial associations under osmotic stress in vitro

World Journal of Microbiology and Biotechnology - Derived from RNA, 5?-ribonucleotides, especially Inosine-5?-monophosphate (IMP) and guanosine-5?-monophosphate (GMP), can enhance...     

In vitro differentiation of myxoid liposarcomas maintained in organ culture system.

Liposarcoma falls into the differential diagnosis of myxoid malignant mesenchymal tumors. On the other hand, its relation with white or brown fat is controversial. Two cases of liposarcoma have been studied by organ culture, a method which provides cell and tissue redifferentiation in vitro. Both cases developed successively cytoplasmic glycogen granules and lipid droplets as well as a single lipidic vacuole in the late phase of cultivation as a marker of fat differentiation. Our results support the possibility of identifying myxoid liposarcomas as well as their origin from white fat tissue.     

Phenytoin induces interleukin-1 production in vitro

Human adherent mononuclear cells and subcloned cell lines established from the human histiocytic cell line U-937 were cultured with phenytoin (PHT) and/or lipopolysaccharide (LPS) purified from Bacteroides fragilis. After the cultivation period, the cell-free supernatants were tested for interleukin-1 (Il-1) activity. The results showed that PHT induces Il-1 activity and potentiates LPS-induced Il-1 production. In the monocytic cell line U-937, the induced Il-1 production was found to be clonally distributed indicating that the response to PHT may be exerted by a subpopulation of monocytes. The PHT-induced Il-1 activity may be of importance in the development of gingival overgrowth. The induced Il-1 could also contribute to other known side effects such as dermatologic reactions accompanied by transient fever seen in patients medicating the drug.     

The ultrastructure of neuromuscular and neural-neural relationships in in vitro maintained Dirofilaria Immitis

Relationships of neuromuscular junctions of the somatic musculature and associated neural-neural synapses in the ventral nerve trunk of the canine adult heartworm, Dirofilaria immitis, were studied by transmission electron microscopy. The heartworms were maintained in vitro prior to study. Nerve fibres in the trunk were highly invaginated into the cytoplasm of hypodermal cells and connected through the intercellular spaces via mesaxons. The nerve fibres contained neurotubules, neurofilaments and ribosomes. The nerve trunk and the muscle arms were separated by an epineurium averaging 250 nm in width. At the junctional site, a marked reduction in width of the epineurium was noted at the synaptic cleft. Often when two adjacent nerve fibres had adjacent neuromuscular junctions, an axo-axonal synapse and common mesaxon between the adjacent fibres were present. Varicosities were evident on some cross-sections through nerve fibres and ranged from a simple outward swelling against the muscle arm mass to exaggerated outgrowths measuring several micrometers in length.     

Transmission electron microscopic observations on lipid release in Schistosoma mansoni maintained in vitro

Transmission electron microscopy was done on Schistosoma mansoni adult males and females unpaired immediately following recovery from mice and on worms maintained in Earle's balanced salt solution (EBSS) containing 0.1% glucose and 0.5% lactalbumin hydrolysate for 1.0 h at 37°C. Males incubated for 0.5 and 1.0 h released lipids from non-tuberculate and tuberculate areas of the dorsal tegument. Lipid release was not seen in females. Transmission electron microscopy revealed no apparent damage in worms maintained in BBSS for 1.0 h. Our observations suggest that structures described as “spheres” (Weisberg, Carlisle &; Bentley, 1983) are lipid droplets.     

Myostatin induces autophagy in skeletal muscle in vitro

Myostatin is an important regulator of muscle mass that contributes to the loss of muscle mass in a number of chronic diseases. Myostatin is known to activate the expression of components of the ubiquitin-proteosomal pathway but its effect on the autophagic pathway is not known. We therefore analysed the effect of myostatin and TGF-β on autophagy in C2C12 cells by determining the effect of these proteins on LC3 processing, autophagosome formation and autophagy gene expression. Both myostatin and TGF-β increased LC3II expression and turnover as well as autophagosome formation (marked by the formation of puncta in LC3-GFP transfected cells). Myostatin also significantly increased the expression of ATG-4B and ULK-2 mRNA while TGF-β caused a trend towards an increase in these genes. We conclude that myostatin and TGF-β increase autophagy in skeletal muscle cells.     

Streptozotocin induces lipolysis in rat adipocytes in vitro

Streptozotocin (STZ) is used to induce experimental diabetes in animals and is also applied for the treatment of patients with insulinoma. The aim of the present work was to investigate the direct effect of STZ on lipolysis in isolated rat adipocytes. After the isolation, the cells were incubated in a Krebs-Ringer buffer of pH 7.4, at the temperature 37 degrees C for 90 min with different concentrations of STZ: 0.5, 1 or 2 mmol/l. STZ caused a significant rise in basal values (99%, 199%, and 377%, respectively) and epinephrine-stimulated (1 micromol/l) lipolysis (15%, 24% and 46%, respectively). Augmentation of basal lipolysis by STZ was neither restricted by insulin (1 nmol/l) nor by H-89 (an inhibitor of protein kinase A, 50 micromol/l). These results indicate the stimulatory influence of STZ on the action of hormone-sensitive lipase in isolated cells of white adipose tissue. The obtained outcomes suggest that in studies employing STZ, it is necessary to consider its direct effect upon lipolysis in adipocytes.     

Artificial associations between Daucus and nitrogen-fixing Azotobacter cells in vitro

Symbiotic associations were established between nitrogen-fixing Azotobacter zettuovii (CRS-H6) cells and carrot ( Daucus carota L. cv. 'Rother' Half Long) tissues based on the induced carbon and energy dependency of diazotrophs on plant metabolic activity. Symbiotic associations were grown on nitrogen-free media for four years. Plant regeneration was achieved from callus-bacterium associations on nitrogen-containing media. Light and electron micrographs are used to show the localization of bacteria in intercellular spaces of callus and regenerated plant tissues. The nitrogen-fixing ability of the partnership was proved on nitrogen-free media.