Why is ctp important

There is a legal requirement for all motor vehicles used on roads or road-related areas to be registered. Trailers do not need a Greenslip. Before you register your car, it is necessary to know the importance of CTP Greenslips and buy Compulsory Third Party Insurance CTP , or a Greenslip as it is also referred to in New South Wales, and this covers your liability if you or someone who is driving your car causes personal injury to someone else, including your passengers, in an accident.

If you do not have CTP insurance and your vehicle is not registered, you may be personally liable for any injuries you cause and other penalties may apply which can run into many thousands of dollars. One should also note that if you have a valid Greenslip and your car is not registered, then your Greenslip offers no protection as it is not active through your vehicle registration. States also vary on the basis of other factors, including fault, liability, injury and compensation.

In addition, states also have different requirements for vehicle safety and identity checks to be satisfied before registration. Learn more about the rules and requirements for compulsory third party insurance and registration in the state where you live:. Australian Capital Territory. Northern Territory. New South Wales. South Australia.

Western Australia. Regardless of the state, CTP insurance will not cover you for damage to vehicles yours or other parties , damage to property or theft. You need to insure separately for these losses. Compulsory third party insurance provides for compensation for people injured or killed when a vehicle is involved in an accident. Effectively, the driver of the vehicle causing the injury is being indemnified against claims for the damage and losses that were caused.

Third party personal insurance is compulsory so compensation for loss is not dependent on the means of the person who caused the loss. Without CTP insurance, compensation may not be available equally to all parties who are entitled to some form of compensation. CTP synthase is regulated at the transcriptional level Wylie et al. The assembly of CTP synthase into cytoophidia has been proposed to be an additional mechanism of regulation, in which the enzymatic activity is controlled by storing the protein in polymers composed of inactive or less active protein in E.

Nevertheless, filaments containing active CTP synthase have also been proposed for Drosophila Strochlic et al. Recently, Lynch et al. In contrast, they found a different architecture for filaments composed of human CTP synthase 1, where the protein was present in an active conformation Lynch et al.

Although Tg CTPS is highly expressed in extracellular as well as in intracellular tachyzoites, our data show that filament-like structure occur in a stage-dependent manner in T. We hypothesize that when the parasite is outside the host cell, it requires a low CTPS activity, likely regulated by protein assembly into filaments acting as the transient enzyme storage. In contrast, intracellular parasites replicating within vacuoles should require a congruently high CTPS activity to satisfy the nucleotide and lipid biogenesis.

This is plausibly controlled by the release of CTPS from filament-like structures. Our future work will involve the live imaging to follow dynamic changes in the spatial expression of Tg CTPS during the invasion and egress of tachyzoites.

The dynamics of CTP synthase filament in organisms where it has been studied appears to be affected by the cell cycle, nutrient deprivation, all four nucleotides, and enzyme inhibitors. In contrast, high levels of CTP stimulate the filament assembly in E. It is interesting to note that glucose and glutamine serve as two major sources of carbon for the parasite Blume et al. Further experiments involving the parasite mutants defective in glucose and glutamine catabolism are required to determine whether the increase in CTPS structures is indeed related to the sensing of these nutrients.

It has been studied as an anticancer drug in several Phase II clinical trials in the s, because tumor cells are more susceptible to the glutamine analogs compared to normal cells. Its therapeutic usage has been abandoned due its tendency to cause nausea Cervantes-Madrid et al. The inhibitor has divergent effects on the localization of different CTPS enzymes. Analogs of glutamine such as azaserine, acivicin and DON exhibit strong anti-parasitic activities against Plasmodium, Trypanosoma and Toxoplasma Jaffe, ; Hofer et al.

Tachyzoites treated with DON were aberrantly shaped and apparently defective in endodyogeny. These results together signify a potential therapeutic repurposing of this drug to treat toxoplasmosis. Two possible scenarios could explain the relationship of CTPS to morphological alterations observed in intracellular and extracellular parasites. Gupta and co-workers observed that an analog of choline interferes with the synthesis of phosphatidylcholine, the most abundant glycerophospholipid in the parasite.

It results in a dramatic effect on growth and membrane composition concurrent with an aberrant morphology Gupta et al. In an alternative scenario, the ability of Tg CTPS to form filament-like structures and the inhibitory effect of DON on the parasite replication suggest that this protein could somehow be associated with the inner membrane complex IMC or with other component s of the parasite pellicle, the structure involved in maintaining the parasite shape.

This would be consistent with a cytoskeleton-like function of CTP synthase filaments observed in some bacteria Ingerson-Mahar et al. Our prospective work involves further characterization of filament-like structures by site-directed mutagenesis and electron microscopy to discern the mechanistic roles of CTPS structures.

Tg CTPS activity is therefore likely to be important for acute and chronic infections. In cancer cells, where the proliferation rate is increased compared to normal cells, CTP is obtained mostly via CTP synthase van den Berg et al. In contrast, T. Furthermore, pyrimidine phosphorylase, another salvage enzyme, was unable to use cytidine as a substrate Iltzsch, The parasite's inability to salvage CTP to compensate for the inhibition or absence of Tg CTPS, supports the notion that this is an essential gene for parasite survival.

According to this model, genes are assigned values reflecting their contribution to parasite fitness. The maximum value for a non-essential gene is 2. The physiologically essential nature of Tg CTPS could be exploited to design specific inhibitors of the parasite growth. Figure 8. Pyrimidine salvage and interconversion reactions in T. The numbers next to the arrows represent the enzymes involved in each reaction, which are listed in the table.

The dotted red lines show conversions that probably do not take place in tachyzoites, for the reasons given in the table l. Data were extracted from: 1 ToxoDB www. Reagents were purchased from Sigma-Aldrich unless noted otherwise. Restriction endonucleases, T4 ligase and calf intestinal alkaline phosphatase were from New England Biolabs. Reagents for cell culture were from Biowest. The following primary antibodies were used in western blot or immunofluorescence assays: mouse or rabbit anti c-myc monoclonal antibodies, M and C, Sigma , anti-PentaHis antibody Qiagen , rabbit anti- Tg Hsp90 Echeverria et al.

All transgenic and mutant parasite lines are derivatives of the RH parental strain. Tachyzoites from all strains were propagated in vitro by serial passages in human foreskin fibroblasts HFF host cells, as previously described Jacot et al.

Briefly, parasites were routinely cultured on confluent HFF cell monolayers at a multiplicity of infection MOI of 3, until their complete lysis. The recombinant proteins were expressed in the E. Expression conditions, purification and refolding of insoluble Tg CTPS by matrix assisted method are described in the Supplementary Material.

Assay parameters were fitted with the SkanIt T M software. Ionic strength was adjusted to 0. The values of the equation parameters were: k 0 , 1. Tg CTPS activity was linear with reaction time and dependent on enzyme concentration data not shown.

Kinetic parameters were determined using GraphPad Prism v6e software. Stripping of the membrane was performed using the western blot recycling kit Alpha diagnostics international. After 25—30 h of incubation, the medium was removed, and the infected cells were washed twice with 1x PBS. Subsequently, cells were permeabilized in 0. The coverslips were incubated for 1 h in the primary antibody solution and washed 3 times for 5 min each in 0.

Fluorescent imaging was performed using either an ApoTome Imager. We performed a complementation assay using S. George M. Because ura7 and ura8 are synthetic lethal, the growth of this strain requires the presence of a plasmid expressing a functional CTPS.

In addition, it lacks beta-isopropylmalate dehydrogenase LEU2 that catalyzes the third step in leucine biosynthesis, as well as OMP decarboxylase URA3 catalyzing the sixth step in pyrimidine biosynthesis.

The replacement of this plasmid with a plasmid expressing Tg CTPS plasmid shuffling was done in two steps, as described previously Han et al. The empty vector was used as a negative control. Transformants were selected on plates with SC medium without leucine or uracil Figure 2B , left. These cells were auxotrophic for uracil Figure 2C , right , since the absence of pyrimidine biosynthesis is compensated by uracil salvage. Parasites expressing an ectopic copy of Tg CTPS-c-myc protein were used for further localization experiments.

As in the first construct, the reverse primer included the c-myc tag. The transfected parasites were added to the HFF monolayer cells without selection for 10—24 h.

A clonal stable parasite line was isolated by limiting dilution in well plates. Replication assays were performed to evaluate the ability of T. Confluent HFFs cells were grown on round glass coverslips within well plates and infected with fresh parasites at a MOI of 1.

Three independent biological experiments were performed and the replication rates under different conditions were compared. For each assay, the number of parasites per vacuole was counted for around vacuoles. Plaque assays were used to assess the overall growth fitness of the parasite strain under indicated conditions Black and Boothroyd, HFF cell monolayers in 6-well plates were inoculated with — parasites.

Plates were incubated without perturbation under indicated growth conditions. Three independent biological replicates were done to calculate the mean number and area of at least 50 plaques under each condition. Working dilutions for cell culture were made in DMEM from the stock solution.

For the gene knockdown, a fragment of 1. The attempt to generate a conditional mutant by Cre recombinase activity was performed using a gene-swap strategy Andenmatten et al. The tetracycline regulation of Tg CTPS-c-myc was confirmed by IFA, comparing parasites cultured without anhydrotetracycline ATc versus parasites cultured for 40 h in medium containing 0.

Stable parasites were selected with chloramphenicol as previously described Kim et al. HN-O and AL performed the experiments. All authors reviewed the results and approved the final version of the manuscript. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The research was also supported by funding from the Facultad de Ciencias and the Vicerrectoria de Investigaciones Universidad de los Andes, Colombia.

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