The ever unfolding story of cAMP signaling in trypanosomatids: vive la difference!

Kinetoplastids are unicellular, eukaryotic, flagellated protozoans containing the eponymous kinetoplast. Within this order, the family of trypanosomatids are responsible for some of the most serious human diseases, including Chagas disease (Trypanosoma cruzi), sleeping sickness (Trypanosoma brucei spp.), and leishmaniasis (Leishmania spp). Although cAMP is produced during the life cycle stages of these parasites, its signaling pathways are very different from those of mammals. The absence of G-protein-coupled receptors, the presence of structurally different adenylyl cyclases, the paucity of known cAMP effector proteins and the stringent need for regulation of cAMP in the small kinetoplastid cells all suggest a significantly different biochemical pathway and likely cell biology. However, each of the main kinetoplastid parasites express four class 1-type cyclic nucleotide-specific phosphodiesterases (PDEA-D), which have highly similar catalytic domains to that of human PDEs. To date, only TbrPDEB, expressed as two slightly different isoforms TbrPDEB1 and B2, has been found to be essential when ablated. Although the genomes contain reasonably well conserved genes for catalytic and regulatory domains of protein kinase A, these have been shown to have varied structural and functional roles in the different species. Recent discovery of a role of cAMP/AMP metabolism in a quorum-sensing signaling pathway in T. brucei, and the identification of downstream cAMP Response Proteins (CARPs) whose expression levels correlate with sensitivity to PDE inhibitors, suggests a complex signaling cascade. The interplay between the roles of these novel CARPs and the quorum-sensing signaling pathway on cell division and differentiation makes for intriguing cell biology and a new paradigm in cAMP signal transduction, as well as potential targets for trypanosomatid-specific cAMP pathway-based therapeutics

Trypanosoma brucei bloodstream forms express highly specific and separate transporters for adenine and hypoxanthine; evidence for a new protozoan purine transporter family?

The transport of nucleobases and nucleosides in protozoan parasites is known to be performed by Equilibrative Nucleoside Transporter (ENT) family members, including the extensively studied P1 and P2 nucleoside transporters of T. brucei bloodstream forms. Studies with P2 knockout parasites suggested the existence of as yet uncharacterised purine transport mechanisms in these cells. Here, we deleted several ENT genes, in addition to P2, including an array comprising three genes encoding for high-affinity broad-selectivity nucleobase transporters - the longest multi-gene locus deletion in T. brucei to date. It was verified that none of them appreciably contributed to the transport of hypoxanthine in bloodstream forms grown axenically in HMI-9 medium, which was mainly performed by a previously not described hypoxanthine-specific transporter (HXT1) with a Km of 22 ± 1.7 μM and Vmax of 0.49 ± 0.06 pmol(107 cells)-1s-1. The uptake of adenine was also assessed in the knockout cells and was performed by a highly specific adenine transporter (ADET1) with a Km of 573 ± 62 nM and Vmax of 0.23 ± 0.06 pmol(107 cells)-1 s-1. Neither HXT1 nor ADET1 displayed any affinity for other natural purines or pyrimidines and could not be completely inhibited by hypoxanthine or adenine analogues. These carriers may be the final pieces in the substantial transporter array trypanosomes can employ to fine-tune the uptake of purines from diverse environments during their life cycles, and may be encoded by genes other than those of the ENT family

Pyrimidine biosynthesis is not an essential function for trypanosoma brucei bloodstream forms

<p>Background: African trypanosomes are capable of both pyrimidine biosynthesis and salvage of preformed pyrimidines from the host, but it is unknown whether either process is essential to the parasite.</p> <p>Methodology/Principal Findings: Pyrimidine requirements for growth were investigated using strictly pyrimidine-free media, with or without single added pyrimidine sources. Growth rates of wild-type bloodstream form Trypanosoma brucei brucei were unchanged in pyrimidine-free medium. The essentiality of the de novo pyrimidine biosynthesis pathway was studied by knocking out the PYR6-5 locus that produces a fusion product of orotate phosphoribosyltransferase (OPRT) and Orotidine Monophosphate Decarboxylase (OMPDCase). The pyrimidine auxotroph was dependent on a suitable extracellular pyrimidine source. Pyrimidine starvation was rapidly lethal and non-reversible, causing incomplete DNA content in new cells. The phenotype could be rescued by addition of uracil; supplementation with uridine, 2′deoxyuridine, and cytidine allowed a diminished growth rate and density. PYR6-5−/− trypanosomes were more sensitive to pyrimidine antimetabolites and displayed increased uracil transport rates and uridine phosphorylase activity. Pyrimidine auxotrophs were able to infect mice although the infection developed much more slowly than infection with the parental, prototrophic trypanosome line.</p> <p>Conclusions/Significance: Pyrimidine salvage was not an essential function for bloodstream T. b. brucei. However, trypanosomes lacking de novo pyrimidine biosynthesis are completely dependent on an extracellular pyrimidine source, strongly preferring uracil, and display reduced infectivity. As T. brucei are able to salvage sufficient pyrimidines from the host environment, the pyrimidine biosynthesis pathway is not a viable drug target, although any interruption of pyrimidine supply was lethal.</p&gt

Cloning and characterisation of the Equilibrative Nucleoside Transporter family of Trypanosoma cruzi: ultra-high affinity and selectivity to survive in the intracellular niche

Background: Trypanosoma cruzi, the causative agent of Chagas' disease is unable to synthesise its own purines and relies on salvage from the host. In other protozoa, purine uptake has been shown to be mediated by Equilibrative Nucleoside Transporters (ENTs). Methods: To investigate the functionality of T. cruzi-encoded ENT transporters, its four putative ENT genes (TcrNB1, TcrNB2, TcrNT1 and TcrNT2) were cloned and expressed in genetically adapted Trypanosoma brucei procyclic cells from which the nucleobase transporter locus was deleted. Results: TcrNB1 displayed very high affinity for hypoxanthine (Km 93.8 ± 4.7 nM for) and guanine, and moderate affinity for adenine. TcrNT1 was found to be a high-affinity guanosine/inosine transporter (inosine Km is 1.0 ± 0.03 μM; guanosine Ki is 0.92 ± 0.2 μM). TcrNT2 encoded a high-affinity thymidine transporter (Km = 223.5 ± 7.1 nM) with a clear preference for 2’-deoxypyrimidines. TcrNB2, whose activity could not be determined in our system, could be a low-affinity purine nucleobase transporter, given its sequence and predicted structural similarities to Leishmania major NT4. All 4 transporter genes were highly expressed in the amastigote forms, with much lower expression in the non-dividing stages. Conclusions: The data appear to show that, surprisingly, T. cruzi has a preference for oxopurines over aminopurines and efficiently transports 2′-deoxypyrimidines. The T. cruzi ENTs display exceptionally high substrate affinity as an adaptation to their intracellular localisation. General significance: This study reports the first cloning of T. cruzi purine and pyrimidine transporters, including the first gene encoding a pyrimidine-selective protozoan transporter

Efficiency of free energy calculations of spin lattices by spectral quantum algorithms

Quantum algorithms are well-suited to calculate estimates of the energy spectra for spin lattice systems. These algorithms are based on the efficient calculation of the discrete Fourier components of the density of states. The efficiency of these algorithms in calculating the free energy per spin of general spin lattices to bounded error is examined. We find that the number of Fourier components required to bound the error in the free energy due to the broadening of the density of states scales polynomially with the number of spins in the lattice. However, the precision with which the Fourier components must be calculated is found to be an exponential function of the system size.Comment: 9 pages, 4 figures; corrected typographical and minor mathematical error

Predicted optimum ambient temperatures for broiler chickens to dissipate metabolic heat do not affect performance or improve breast muscle quality

<p></p><p>An experiment was conducted to test the hypothesis that muscle damage in fast-growing broiler chickens is associated with an ambient temperature that does not permit the birds to lose metabolic heat resulting in physiological heat stress and a reduction in meat quality.</p><p>The experiment was performed in 4 climate chambers and was repeated in 2 trials using a total of 200 male broiler chickens. Two treatments compared the recommended temperature profile and a cool regimen. The cool regimen was defined by a theoretical model that determined the environmental temperature that would enable heat generated by the bird to be lost to the environment.</p><p>There were no differences in growth rate or feed intake between the two treatments. Breast muscles from birds on the recommended temperature regimen were lighter, less red and more yellow than those from the cool temperature regimen. There were no differences in moisture loss or shear strength but stiffness was greater in breast muscle from birds housed in the cool compared to the recommended regimen.</p><p>Histopathological changes in the breast muscle were similar in both treatments and were characterised by mild to severe myofibre degeneration and necrosis with regeneration, fibrosis and adipocyte infiltration. There was no difference in plasma creatine kinase activity, a measure of muscle cell damage, between the two treatments consistent with the absence of differences in muscle pathology.</p><p>It was concluded that breast muscle damage in fast-growing broiler chickens was not the result of an inability to lose metabolic heat at recommended ambient temperatures. The results suggest that muscle cell damage and breast meat quality concerns in modern broiler chickens are related to genetic selection for muscle yields and that genetic selection to address breast muscle integrity in a balanced breeding programme is imperative.</p><p></p> <p>An experiment was conducted to test the hypothesis that muscle damage in fast-growing broiler chickens is associated with an ambient temperature that does not permit the birds to lose metabolic heat resulting in physiological heat stress and a reduction in meat quality.</p> <p>The experiment was performed in 4 climate chambers and was repeated in 2 trials using a total of 200 male broiler chickens. Two treatments compared the recommended temperature profile and a cool regimen. The cool regimen was defined by a theoretical model that determined the environmental temperature that would enable heat generated by the bird to be lost to the environment.</p> <p>There were no differences in growth rate or feed intake between the two treatments. Breast muscles from birds on the recommended temperature regimen were lighter, less red and more yellow than those from the cool temperature regimen. There were no differences in moisture loss or shear strength but stiffness was greater in breast muscle from birds housed in the cool compared to the recommended regimen.</p> <p>Histopathological changes in the breast muscle were similar in both treatments and were characterised by mild to severe myofibre degeneration and necrosis with regeneration, fibrosis and adipocyte infiltration. There was no difference in plasma creatine kinase activity, a measure of muscle cell damage, between the two treatments consistent with the absence of differences in muscle pathology.</p> <p>It was concluded that breast muscle damage in fast-growing broiler chickens was not the result of an inability to lose metabolic heat at recommended ambient temperatures. The results suggest that muscle cell damage and breast meat quality concerns in modern broiler chickens are related to genetic selection for muscle yields and that genetic selection to address breast muscle integrity in a balanced breeding programme is imperative.</p

Quantitative trait loci for bone traits segregating independently of those for growth in an F-2 broiler X layer cross

An F broiler-layer cross was phenotyped for 18 skeletal traits at 6, 7 and 9 weeks of age and genotyped with 120 microsatellite markers. Interval mapping identified 61 suggestive and significant QTL on 16 of the 25 linkage groups for 16 traits. Thirty-six additional QTL were identified when the assumption that QTL were fixed in the grandparent lines was relaxed. QTL with large effects on the lengths of the tarsometatarsus, tibia and femur, and the weights of the tibia and femur were identified on GGA4 between 217 and 249 cM. Six QTL for skeletal traits were identified that did not co-locate with genome wide significant QTL for body weight and two body weight QTL did not coincide with skeletal trait QTL. Significant evidence of imprinting was found in ten of the QTL and QTL x sex interactions were identified for 22 traits. Six alleles from the broiler line for weight- and size-related skeletal QTL were positive. Negative alleles for bone quality traits such as tibial dyschondroplasia, leg bowing and tibia twisting generally originated from the layer line suggesting that the allele inherited from the broiler is more protective than the allele originating from the layer

Effects of pressure on diffusion and vacancy formation in MgO from non-empirical free-energy integrations

The free energies of vacancy pair formation and migration in MgO were computed via molecular dynamics using free-energy integrations and a non-empirical ionic model with no adjustable parameters. The intrinsic diffusion constant for MgO was obtained at pressures from 0 to 140 GPa and temperatures from 1000 to 5000 K. Excellent agreement was found with the zero pressure diffusion data within experimental error. The homologous temperature model which relates diffusion to the melting curve describes well our high pressure results within our theoretical framework.Comment: 4 pages, latex, 1 figure, revtex, submitted to PR