Identification of the Toxoplasma gondii mitochondrial ribosome, and characterisation of a protein essential for mitochondrial translation

Apicomplexan parasites cause diseases such as malaria and toxoplasmosis. The apicomplexan mitochondrion shows striking differences from common model organisms, including fundamental processes such as mitochondrial translation. Despite evidence that mitochondrial translation is essential for parasite survival, it is largely understudied. Progress has been restricted by the absence of functional assays to detect apicomplexan mitochondrial translation, a lack of knowledge of proteins involved in the process and the inability to identify and detect mitoribosomes. We report the localization of 12 new mitochondrial proteins, including 6 putative mitoribosomal proteins. We demonstrate the integration of three mitoribosomal proteins in macromolecular complexes, and provide evidence suggesting these are apicomplexan mitoribosomal subunits, detected here for the first time. Finally, a new analytical pipeline detected defects in mitochondrial translation upon depletion of the small subunit protein 35 (TgmS35), while other mitochondrial functions remain unaffected. Our work lays a foundation for the study of apicomplexan mitochondrial translation

Th1/Th2 cytokines and ICAM-1 levels post-liver transplant do not predict early rejection.

Th1 derived cytokines IFN-gamma and IL-2, Th2 cytokine IL-4, and ICAM-1 have been implicated in liver allograft rejection. In order to determine whether monitoring of cytokine profiles during the first days post-liver transplant can predict early rejection we measured IFN-gg, IL-2, sIL-2 receptor, IL-4 and ICAM-1 in 22 patients, in plasma samples obtained within 4 h after liver perfusion (baseline) and between postoperative days (POD) 3-6. ICAM-1 and sIL-2R levels at POD 3-6 were significantly higher than at baseline but did not differ in presence or absence of rejection. Mean percentage increase of ICAM-1 levels was significantly lower in patients with Muromonab-C3 Orthoclone OKT3 (J.C. Health Care) (OKT3) whereas percentage increase of sIL-2R levels was higher in OKT3-treated patients. IFN-gamma levels at POD 3-6 increased from baseline while IL-4 levels were unchanged. Levels of IFN-gamma, IL-4 and their ratios did not correlate with rejection or immunosuppressive therapy. Thus, Th1/Th2 cytokine monitoring during the first week post-transplant does not predict early rejection and immunosuppressive therapy is the predominant factor affecting ICAM and sIL-2R levels after liver transplantation

Actinomyces israelii May Produce Vulvar Lesions Suspicious for Malignancy

Background. We present a case of Actinomyces israelii causing vulvar mass suspicious for malignancy in a postmenopausal woman. Case. A 60 year-old woman presented due to a firm, nonmobile, 10 cm vulvar mass, which had been rapidly enlarging for 5 months. The mass was painful, with localized pruritus and sinus tracts oozing of serosanguinous fluid. Biopsy and cultures revealed a ruptured epidermal inclusion cyst containing granulation tissue and Actinomyces israelii. Conclusion. Actinomyces israelii may produce vulvar lesions that are suspicious for malignancy. Thus, biopsies and cultures are both mandatory while evaluating vulvar masses suspicious for malignancy

Recurrence of hepatitis C after liver transplantation is associated with increased systemic IL-10 levels.

BACKGROUND: Recurrence of hepatitis C after liver transplantation is an almost universal occurrence. T-cell derived cytokines have an important role in the development of liver damage associated with chronic hepatitis C, their post-transplant levels, however, have not been correlated with histologic recurrence of the disease. AIMS: We sought to analyze levels of TNF-alpha, soluble IL-2 receptor, IL-4 and IL-10 at 1 month, 6 months and 1 year after transplantation in 27 patients undergoing transplantation for hepatitis C related end-stage liver disease. METHODS: HCV RNA levels were monitored by a branched-chain DNA signal amplification assay. Diagnosis of recurrent hepatitis was based on 1-year protocol biopsies and on biopsies performed for liver enzyme elevations. RESULTS: Recurrent hepatitis C was detected in 52% (n=14) of the 27 patients. HCV RNA levels rose over time in all patients regardless of histologic recurrence. TNF-alpha, and IL-4 levels, although elevated, did not show specific patterns over time or in correlation with recurrence. Similarly, the early elevation followed by a gradual decrease over the first year in the amount of soluble IL-2 receptor was not related to histologic recurrence. We observed a significant increase in circulating IL-10 levels over the first year in patients with biopsy-proven recurrence, while patients with no signs of histologic recurrence displayed increased, but steady levels. CONCLUSIONS: These results suggest that while these cytokines are associated with post-transplant recurrence of hepatitis C, their production may be altered by additional factors

Functional and biochemical characterization of the Toxoplasma gondii succinate dehydrogenase complex

The mitochondrial electron transport chain (mETC) is a series of membrane embedded enzymatic complexes critical for energy conversion and mitochondrial metabolism. In commonly studied eukaryotes, including humans and animals, complex II, also known as succinate dehydrogenase (SDH), is an essential four-subunit enzyme that acts as an entry point to the mETC, by harvesting electrons from the TCA cycle. Apicomplexa are pathogenic parasites with significant impact on human and animal health. The phylum includes Toxoplasma gondii which can cause fatal infections in immunocompromised people. Most apicomplexans, including Toxoplasma, rely on their mETC for survival, yet SDH remains largely understudied. Previous studies pointed to a divergent apicomplexan SDH with nine subunits proposed for the Toxoplasma complex, compared to four in humans. While two of the nine are homologs of the well-studied SDHA and B, the other seven have no homologs in SDHs of other systems. Moreover, SDHC and D, that anchor SDH to the membrane and participate in substrate bindings, have no homologs in Apicomplexa. Here, we validated five of the seven proposed subunits as bona fide SDH components and demonstrated their importance for SDH assembly and activity. We further find that all five subunits are important for parasite growth, and that disruption of SDH impairs mitochondrial respiration and results in spontaneous initiation of differentiation into bradyzoites. Finally, we provide evidence that the five subunits are membrane bound, consistent with their potential role in membrane anchoring, and we demonstrate that a DY motif in one of them, SDH10, is essential for complex formation and function. Our study confirms the divergent composition of Toxoplasma SDH compared to human, and starts exploring the role of the lineage-specific subunits in SDH function, paving the way for future mechanistic studies

The mystery of massive mitochondrial complexes: the apicomplexan respiratory chain

The mitochondrial respiratory chain is an essential pathway in most studied eukaryotes due to its roles in respiration and other pathways that depend on mitochondrial membrane potential. Apicomplexans are unicellular eukaryotes whose members have an impact on global health. The respiratory chain is a drug target for some members of this group, notably the malaria-causing Plasmodium spp. This has motivated studies of the respiratory chain in apicomplexan parasites, primarily Toxoplasma gondii and Plasmodium spp. for which experimental tools are most advanced. Studies of the respiratory complexes in these organisms revealed numerous novel features, including expansion of complex size. The divergence of apicomplexan mitochondria from commonly studied models highlights the diversity of mitochondrial form and function across eukaryotic life

Complexome profile of Toxoplasma gondii mitochondria identifies divergent subunits of respiratory chain complexes including new subunits of cytochrome bc1 complex.

The mitochondrial electron transport chain (mETC) and F1Fo-ATP synthase are of central importance for energy and metabolism in eukaryotic cells. The Apicomplexa, important pathogens of humans causing diseases such as toxoplasmosis and malaria, depend on their mETC in every known stage of their complicated life cycles. Here, using a complexome profiling proteomic approach, we have characterised the Toxoplasma mETC complexes and F1Fo-ATP synthase. We identified and assigned 60 proteins to complexes II, IV and F1Fo-ATP synthase of Toxoplasma, of which 16 have not been identified previously. Notably, our complexome profile elucidates the composition of the Toxoplasma complex III, the target of clinically used drugs such as atovaquone. We identified two new homologous subunits and two new parasite-specific subunits, one of which is broadly conserved in myzozoans. We demonstrate all four proteins are essential for complex III stability and parasite growth, and show their depletion leads to decreased mitochondrial potential, supporting their assignment as complex III subunits. Our study highlights the divergent subunit composition of the apicomplexan mETC and F1Fo-ATP synthase complexes and sets the stage for future structural and drug discovery studies

Nonlinear pharmacokinetics of therapeutic proteins resulting from receptor mediated endocytosis

Receptor mediated endocytosis (RME) plays a major role in the disposition of therapeutic protein drugs in the body. It is suspected to be a major source of nonlinear pharmacokinetic behavior observed in clinical pharmacokinetic data. So far, mostly empirical or semi-mechanistic approaches have been used to represent RME. A thorough understanding of the impact of the properties of the drug and of the receptor system on the resulting nonlinear disposition is still missing, as is how to best represent RME in pharmacokinetic models. In this article, we present a detailed mechanistic model of RME that explicitly takes into account receptor binding and trafficking inside the cell and that is used to derive reduced models of RME which retain a mechanistic interpretation. We find that RME can be described by an extended Michaelis–Menten model that accounts for both the distribution and the elimination aspect of RME. If the amount of drug in the receptor system is negligible a standard Michaelis–Menten model is capable of describing the elimination by RME. Notably, a receptor system can efficiently eliminate drug from the extracellular space even if the total number of receptors is small. We find that drug elimination by RME can result in substantial nonlinear pharmacokinetics. The extent of nonlinearity is higher for drug/receptor systems with higher receptor availability at the membrane, or faster internalization and degradation of extracellular drug. Our approach is exemplified for the epidermal growth factor receptor system

Risks of pregnancy and birth in obese primiparous women: an analysis of German perinatal statistics

PURPOSE: To compare risks of pregnancy and birth in obese (body mass index, BMI ≥ 30) and normal weight women (BMI 18.5–24.99) giving birth to their first child. METHODS: We analysed data of 243,571 pregnancies in primiparous women from the German perinatal statistics of 1998–2000. We calculated odds ratios (ORs) with 95% confidence intervals (CIs) for selected pregnancy and birth risks. ORs were adjusted for the confounding factors age, smoking status, single mother status, and maternal education. RESULTS: Obesity during pregnancy is common in primiparous women (n = 19,130; 7.9% of all cases) and it is significantly associated with a number of risks of pregnancy and birth, including diabetes [OR 3.71 (95% CI 2.93; 4.71); p < 0.001], hypertension [OR 8.44 (7.91; 9.00); p < 0.001], preecalmpsia/eclampsia [OR 6.72 (6.30; 7.17); p < 0.001], intraamniotic infection [OR 2.33 (2.05; 2.64); p < 0.001], birth weight ≥4,000 g [OR 2.16 (2.05; 2.28); p < 0.001], and an increased rate of Caesarean section [OR 2.23 (2.15; 2.30); p < 0.001]. Some risks were less frequent in the obese such as cervical incompetence [OR 0.55 (0.48; 0.63); p < 0.001] and preterm labour [OR 0.47 (0.43; 0.51); p < 0.001]. CONCLUSIONS: Obesity during pregnancy is an important clinical problem in primiparous women because it is common and it is associated with a number of risks of pregnancy and birth. Because of these increased risks, obese women need special attention clinically during the course of their first pregnancy. Weight reduction before the first pregnancy is generally indicated in obese women to prevent the above-mentioned complications of pregnancy and birth