Regulated cleavage of intracellular glycosylphosphatidylinositol in a trypanosome. Peroxisome-to-endoplasmic reticulum translocation of a phospholipase C

Cell exposure to hypo-osmolarity and alkalinity triggers a spectrum of responses including activation of phospholipases. Glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) is expressed in Trypanosoma brucei, a protozoan parasite that causes human African trypanosomiasis. We examined possible contributions of GPI-PLC to the response of T. brucei to hypo-osmotic or mildly alkaline conditions. GPIs were detected at the endoplasmic reticulum (ER). They were cleaved after exposure of T. brucei to hypo-osmolarity or mild alkalinity, which also, strikingly, caused translocation of GPI-PLC from glycosomes (peroxisomes) to the ER. A catalytically inactive Gln81Leu mutant of GPI-PLC failed to cleave GPIs despite being transported from glycosomes to the ER after hypo-osmotic or mild alkaline treatment of the parasites. In contrast, a Cys347Ser mutant of the enzyme could not exit glycosomes after treatment of cells expressing the protein with mild base or hypo-osmotic buffer. We conclude that: (a) GPI-PLC contributes to loss of GPIs in T. brucei treated with hypo-osmotic or mildly alkaline buffer; (b) access of GPI-PLC to its substrate in vivo can be regulated post-translationally; (c) translocation of GPI-PLC from glycosomes to the ER is important for in vivo cleavage of GPIs; (d) Cys347 is part of a peptide motif required for post-translational targeting of GPI-PLC to the ER. Glycosome-to-ER movement of GPI-PLC reveals a novel pathway for intracellular protein traffic. The physiological significance of GPI digestion in cells exposed to mildly alkalinity or hypo-osmolarity is discussed.     

A critical evaluation of Brazilian life cycle assessment studies

Purpose

A critical evaluation of the life cycle assessment (LCA) studies was performed in the main scientific bibliographic databases (online and free access) of Brazil where the LCA methodology could be considered.

Methods

This has been an exploratory study with a qualitative evaluation of quantitative LCA studies with regard to International Organization of Standardization (ISO) 14040 standards. Firstly, the selected papers were those which used the LCA methodology in case studies (quantitative LCA studies). This survey was based on previously chosen keywords which were directly and/or indirectly related to LCA in Portuguese, English, and Spanish.

Results and discussion

One hundred and twenty papers related to LCA were found, among which 21 have been effectively used the LCA methodology applied to case studies. The study has indicated agriculture and livestock as some promising areas for the use of LCA methodology in Brazil. As for the scope of LCA, it has been found that nine papers have adopted the cradle-to-grave approach, whereas 12 papers have limited the study to some life cycle stage (cradle-to-gate, gate-to-gate, or gate-to-grave). This behavior can be justified by the difficulty in obtaining data from raw material, supply chain, inputs, or about the disposal, reuse, and recycling of products/systems. The criteria set out in the ISO 14040 standard was carried out in 17 out of the 21 selected papers.

Conclusions

The LCA of Brazilian studies could be improved. For instance, when considering the requirements and guidelines of ISO standards, at the goal phase, the papers have clearly mentioned their target audience. The scope phase requires more explanation about the allocation procedures, once the process/product is not isolated, and for most processes, it may generate more than one product. As regards the Life Cycle Inventory, these studies could improve their data sources, once few papers used primary sources. According to our understanding, the best phase performed by the papers was life cycle impact assessment. Hopefully, LCA will become a known research area and will be adopted by most of the Brazilian scientific community. It is further expected that LCA might have a regular publication in scientific journals (perhaps an own journal).     

The cell cycle modulated glycoprotein GP115 is one of the major yeast proteins containing glycosylphosphatidylinositol

The cell cycle modulated protein gp115 (115 kDa, isoelectric point about 4.8-5) of Saccharomyces cerevisiae undergoes various post-translational modifications. It is N-glycosylated during its maturation along the secretory pathway where an intermediary precursor of 100 kDa (p100), dynamically related to the mature gp115 protein, is detected at the level of endoplasmic reticulum. Moreover, we have shown by the use of metabolic labeling with [35S]methionine, [3H]palmitic acid and myo-[3H]inositol combined with high resolution two-dimensional gel electrophoresis and immunoprecipitation with a specific antiserum, that gp115 is one of the major palmitate- and inositol-containing proteins in yeast. These results, and the susceptibility of gp115 to phosphatidylinositol-specific phospholipase C treatment strongly indicate that gp115 contains the glycosylphosphatidylinositol (GPI) structure as membrane anchor domain. The two-dimensional analysis of the palmitate- and inositol-labeled proteins has also allowed the characterization of other polypeptides which possibly contain a GPI structure.     

A spatially explicit life cycle inventory of the global textile chain

Background, aim, and scope  Life cycle analyses (LCA) approaches require adaptation to reflect the increasing delocalization of production to emerging countries. This work addresses this challenge by establishing a country-level, spatially explicit life cycle inventory (LCI). This study comprises three separate dimensions. The first dimension is spatial: processes and emissions are allocated to the country in which they take place and modeled to take into account local factors. Emerging economies China and India are the location of production, the consumption occurs in Germany, an Organisation for Economic Cooperation and Development country. The second dimension is the product level: we consider two distinct textile garments, a cotton T-shirt and a polyester jacket, in order to highlight potential differences in the production and use phases. The third dimension is the inventory composition: we track CO₂, SO₂, NO _x , and particulates, four major atmospheric pollutants, as well as energy use. This third dimension enriches the analysis of the spatial differentiation (first dimension) and distinct products (second dimension). Materials and methods  We describe the textile production and use processes and define a functional unit for a garment. We then model important processes using a hierarchy of preferential data sources. We place special emphasis on the modeling of the principal local energy processes: electricity and transport in emerging countries. Results  The spatially explicit inventory is disaggregated by country of location of the emissions and analyzed according to the dimensions of the study: location, product, and pollutant. The inventory shows striking differences between the two products considered as well as between the different pollutants considered. For the T-shirt, over 70% of the energy use and CO₂ emissions occur in the consuming country, whereas for the jacket, more than 70% occur in the producing country. This reversal of proportions is due to differences in the use phase of the garments. For SO₂, in contrast, over two thirds of the emissions occur in the country of production for both T-shirt and jacket. The difference in emission patterns between CO₂ and SO₂ is due to local electricity processes, justifying our emphasis on local energy infrastructure. Discussion  The complexity of considering differences in location, product, and pollutant is rewarded by a much richer understanding of a global production–consumption chain. The inclusion of two different products in the LCI highlights the importance of the definition of a product's functional unit in the analysis and implications of results. Several use-phase scenarios demonstrate the importance of consumer behavior over equipment efficiency. The spatial emission patterns of the different pollutants allow us to understand the role of various energy infrastructure elements. The emission patterns furthermore inform the debate on the Environmental Kuznets Curve, which applies only to pollutants which can be easily filtered and does not take into account the effects of production displacement. We also discuss the appropriateness and limitations of applying the LCA methodology in a global context, especially in developing countries. Conclusions  Our spatial LCI method yields important insights in the quantity and pattern of emissions due to different product life cycle stages, dependent on the local technology, emphasizing the importance of consumer behavior. From a life cycle perspective, consumer education promoting air-drying and cool washing is more important than efficient appliances. Recommendations and perspectives  Spatial LCI with country-specific data is a promising method, necessary for the challenges of globalized production–consumption chains. We recommend inventory reporting of final energy forms, such as electricity, and modular LCA databases, which would allow the easy modification of underlying energy infrastructure. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Hotspot identification in the Indonesian tempeh supply chain using life cycle assessment

The International Journal of Life Cycle Assessment - Tempeh is a traditional Indonesian fermented soybean product, which plays an essential role in its culture and economy and forms an important...     

Ketoacyl synthase domain is a major determinant for fatty acyl chain length in Saccharomyces cerevisiae

Yeast fatty acid synthase (Fas) comprises two subunits, α₆ and β₆, encoded by FAS2 and FAS1, respectively. To determine features of yeast Fas that control fatty acyl chain length, chimeric genes were constructed by combining FAS sequences from Saccharomyces cerevisiae (ScFAS) and Hansenula polymorpha (HpFAS), which mostly produces C16 and C18 fatty acids, respectively. The C16/C18 ratios decreased from 2.2 ± 0.1 in wild-type S. cerevisiae to 1.0 ± 0.1, 0.5 ± 0.2 and 0.8 ± 0.1 by replacement of ScFAS1, ScFAS2 and ScFAS1 ScFAS2 with HpFAS1, HpFAS2 and HpFAS1 HpFAS2, respectively, suggesting that the α, but not β subunits play a major role in determining fatty acyl chain length. Replacement of phosphopantetheinyl transferase (PPT) domain with the equivalent region from HpFAS2 did not affect C16/C18 ratio. Chimeric Fas2 containing half N-terminal ScFas2 and half C-terminal HpFas2 carrying H. polymorpha ketoacyl synthase (KS) and PPT gave a remarkable decrease in C16/C18 ratio (0.6 ± 0.1), indicating that KS plays a major role in determining chain length.     

The Hsp90–Sti1 interaction is critical for Leishmania donovani proliferation in both life cycle stages

The heat shock protein 90 plays a pivotal role in the life cycle control of Leishmania donovani promoting the fast‐growing insect stage of this parasite. Equally important for insect stage growth is the co‐chaperone Sti1. We show that replacement of Sti1 is only feasible in the presence of additional Sti1 transgenes indicating an essential role. To better understand the impact of Sti1 and its interaction with Hsp90, we performed a mutational analysis of Hsp90. We established that a single amino acid exchange in the Leishmania Hsp90 renders that protein resistant to the inhibitor radicicol (RAD), yet does not interfere with its functionality. Based on this RAD‐resistant Hsp90, we established a combined chemical knockout/gene complementation (CKC) approach. We can show that Hsp90 function is required in both insect and mammalian life stages and that the Sti1‐binding motif of Hsp90 is crucial for proliferation of insect and mammalian stages of the parasite. The Sti1‐binding motif in Leishmania Hsp90 is suboptimal – optimizing the motif increased initial intracellular proliferation underscoring the importance of the Hsp90–Sti1 interaction for this important parasitic protozoan. The CKC strategy we developed will allow the future analysis of more Hsp90 domains and motifs in parasite viability and infectivity.     

Benchmarking environmental performance of electric insulator supply chain in India using life cycle assessment

Purpose

This study aims at finding the environmental impacts generated by an electric disk insulator supply chain, used for the distribution of electricity by an open wire system, through a case study. This study also aims at benchmarking the environmental impacts of an electric insulator manufacturing process by taking ideal condition of zero waste as reference.

Methods

Cradle-to-grave life cycle assessment (LCA) has been carried out by following the guidelines provided in ISO 14040 series standards and using Umberto NXT software. ReCiPe endpoint and ReCiPe midpoint impact assessment methodologies have been used to calculate environmental impacts under various categories. The primary data has been collected from a medium-scale manufacturer of electric disk insulators located at Bikaner in north-west India. The secondary data has been taken from ecoinvent 3.0 database and literature. The environmental impacts using endpoint assessment (ecosystem quality, human health, and resources) and midpoint assessment (climate change, fossil depletion, human toxicity, metal depletion, ozone depletion, terrestrial acidification, and water depletion) categories have been computed. Finally, the results are compared and benchmarked against the ideal zero waste condition using three different production scenarios. The limitation of this study is that the data has been collected only from one manufacturer and its supply chain.

Results and discussion

It has been found that the use of steel, electricity, and fuel; transportation of product; and disposal of water generate high environmental impacts in the supply chain. It has also been found that in the electric disk insulator supply chain, the raw material extraction phase has the highest environmental impacts followed by manufacturing, disposal, transportation, and installation phases. This study has also found that benchmark scenario “B” (zero waste condition) is environmentally more efficient in comparison to scenario “A” (actual recycling condition) and scenario “C” (maximum waste condition).

Conclusions

This study has identified that raw materials, resources, and processes in the supply chain of an electric disk insulator manufacturing unit are responsible for the environmental damage. The various manufacturing processes and installation of the electric disk insulators are similar for all manufacturers except the machinery efficiency and the generated waste. This study provides environmental impacts associated with an electric disk insulator manufacturing process under zero waste or ideal conditions (scenario B). These results are used as a benchmark to compare environmental performance of electric disk insulator supply chain operating under actual conditions.

     

Rab14 is critical for maintenance of Mycobacterium tuberculosis phagosome maturation arrest

Mycobacterium tuberculosis arrests phagosomal maturation in infected macrophage, and, apart from health significance, provides a superb model system to dissect the phagolysosomal biogenesis pathway. Here, we demonstrate a critical role for the small GTPase Rab14 in maintaining mycobacterial phagosome maturation block. Four-dimensional microscopy showed that phagosomes containing live mycobacteria accumulated Rab14 following phagocytosis. The recruitment of Rab14 had strong functional consequence, as a knockdown of endogenous Rab14 by siRNA or overexpression of Rab14 dominant-negative mutants (Rab14S25N and Rab14N125I) released the maturation block and allowed phagosomes harboring live mycobacteria to progress into phagolysosomes. Conversely, overexpression of the wild-type Rab14 and the constitutively active mutant Rab14Q70L prevented phagosomes with dead mycobacteria from undergoing default maturation into phagolysosomal organelles. Mechanistic studies demonstrated a role for Rab14 in stimulating organellar fusion between phagosomes and early endosomes but not with late endosomes. Rab14 enables mycobacterial phagosomes to maintain early endosomal characteristics and avoid late endosomal/lysosomal degradative components.     

Relaxed acyl chain specificity of Bordetella UDP-N-acetylglucosamine acyltransferases

Lipid A (endotoxin) is a major structural component of Gram-negative outer membranes. It also serves as the hydrophobic anchor of lipopolysaccharide and is a potent activator of the innate immune response. Lipid A molecules from the genus Bordetella are reported to exhibit unusual structural asymmetry with respect to the acyl chains at the 3- and 3'-positions. These acyl chains are attached by UDP-N-acetylglucosamine acyltransferase (LpxA). To determine the origin of the acyl variability, the single lpxA ortholog present in each of the genomes of Bordetella bronchiseptica (lpxA(Br)), Bordetella parapertussis (lpxA(Pa)), and Bordetella pertussis (lpxA(Pe)) was cloned and expressed in Escherichia coli. In contrast to all LpxA proteins studied to date, LpxA(Br) and LpxA(Pe) display relaxed acyl chain length specificity in vitro, utilizing C(10)OH-ACP, C(12)OH-ACP, and C(14)OH-ACP at similar rates. Furthermore, hybrid lipid A molecules synthesized at 42 degrees C by an E. coli lpxA mutant complemented with lpxA(Pe) contain C(10)OH, C(12)OH, and C(14)OH at both the 3- and 3'-positions, as determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In contrast, LpxA from B. parapertussis did not display relaxed specificity but was selective for C(10)OH-ACP. This study provides an enzymatic explanation for some of the unusual acyl chain variations found in Bordetella lipid A.     

Babesia canis: the life cycle and laboratory maintenance in its arthropod and mammalian hosts

The life cycle of Babesia canis in its arthropod and mammalian hosts, based on extensive personal observation, is described and reviewed. The following are dealt with: signs and symptoms of babesiosis in dogs; pathology; collection, maintenance and breeding of the tick intermediate hosts—Rhipicephalus sanguineus and Haemaphysalis leachi; transmission through tick bites; forms of B. canis in tick ova, larvae, nymphs and adults.     

Lipid-dependent subcellular relocalization of the acyl chain desaturase in yeast

The degree of acyl chain desaturation of membrane lipids is a critical determinant of membrane fluidity. Temperature-sensitive mutants of the single essential acyl chain desaturase, Ole1p, of yeast have previously been isolated in screens for mitochondrial inheritance mutants (Stewart, L.C. and Yaffe, M.P. (1991). J. Cell Biol. 115, 1249-1257). We now report that the mutant desaturase relocalizes from its uniform ER distribution to a more punctuate localization at the cell periphery upon inactivation of the enzyme. This relocalization takes place within minutes at nonpermissive conditions, a time scale at which mitochondrial morphology and inheritance is not yet affected. Relocalization of the desaturase is fully reversible and does not affect the steady state localization of other ER resident proteins or the kinetic and fidelity of the secretory pathway, indicating a high degree of selectivity for the desaturase. Relocalization of the desaturase is energy independent but is lipid dependent because it is rescued by supplementation with unsaturated fatty acids. Relocalization of the desaturase is also observed in cells treated with inhibitors of the enzyme, indicating that it is independent of temperature-induced alterations of the enzyme. In the absence of desaturase function, lipid synthesis continues, resulting in the generation of lipids with saturated acyl chains. A model is discussed in which the accumulation of saturated lipids in a microdomain around the desaturase could induce the observed segregation and relocalization of the enzyme.     

A novel selection regime for differentiation defects demonstrates an essential role for the stumpy form in the life cycle of the African trypanosome

A novel selection scheme has been developed to isolate bloodstream forms of Trypanosoma brucei, which are defective in their ability to differentiate to the procyclic stage. Detailed characterization of one selected cell line (defective in differentiation clone 1 [DiD-1]) has demonstrated that these cells are indistinguishable from the wild-type population in terms of their morphology, cell cycle progression, and biochemical characteristics but are defective in their ability to initiate differentiation to the procyclic form. Although a small proportion of DiD-1 cells remain able to transform, deletion of the genes for glycophosphatidyl inositol-phospholipase C demonstrated that this enzyme was not responsible for this inefficient differentiation. However, the attenuated growth of the Delta-glycophosphatidyl inositol-phospholipase C DiD-1 cells in mice permitted the expression of stumpy characteristics in this previously monomorphic cell line, and concomitantly their ability to differentiate efficiently was restored. Our results indicate that monomorphic cells retain expression of a characteristic of the stumpy form essential for differentiation, and that this is reduced in the defective cells. This approach provides a new route to dissection of the cytological and molecular basis of life cycle progression in the African trypanosome.     

Hemoglobin is a co-factor of human trypanosome lytic factor

Trypanosome lytic factor (TLF) is a high-density lipoprotein (HDL) subclass providing innate protection to humans against infection by the protozoan parasite Trypanosoma brucei brucei. Two primate-specific plasma proteins, haptoglobin-related protein (Hpr) and apolipoprotein L-1 (ApoL-1), have been proposed to kill T. b. brucei both singularly or when co-assembled into the same HDL. To better understand the mechanism of T. b. brucei killing by TLF, the protein composition of TLF was investigated using a gentle immunoaffinity purification technique that avoids the loss of weakly associated proteins. HDL particles recovered by immunoaffinity absorption, with either anti-Hpr or anti-ApoL-1, were identical in protein composition and specific activity for T. b. brucei killing. Here, we show that TLF-bound Hpr strongly binds Hb and that addition of Hb stimulates TLF killing of T. b. brucei by increasing the affinity of TLF for its receptor, and by inducing Fenton chemistry within the trypanosome lysosome. These findings suggest that TLF in uninfected humans may be inactive against T. b. brucei prior to initiation of infection. We propose that infection of humans by T. b. brucei causes hemolysis that triggers the activation of TLF by the formation of Hpr-Hb complexes, leading to enhanced binding, trypanolytic activity, and clearance of parasites.     

Biogenesis of the trypanosome endo-exocytotic organelle is cytoskeleton mediated

Trypanosoma brucei is a protozoan parasite that is used as a model organism to study such biological phenomena as gene expression, protein trafficking, and cytoskeletal biogenesis. In T. brucei, endocytosis and exocytosis occur exclusively through a sequestered organelle called the flagellar pocket (FP), an invagination of the pellicular membrane. The pocket is the sole site for specific receptors thus maintaining them inaccessible to components of the innate immune system of the mammalian host. The FP is also responsible for the sorting of protective parasite glycoproteins targeted to, or recycling from, the pellicular membrane, and for the removal of host antibodies from the cell surface. Here, we describe the first characterisation of a flagellar pocket cytoskeletal protein, BILBO1. BILBO1 functions to form a cytoskeleton framework upon which the FP is made and which is also required and essential for FP biogenesis and cell survival. Remarkably, RNA interference (RNAi)-mediated ablation of BILBO1 in insect procyclic-form parasites prevents FP biogenesis and induces vesicle accumulation, Golgi swelling, the aberrant repositioning of the new flagellum, and cell death. Cultured bloodstream-form parasites are also nonviable when subjected to BILBO1 RNAi. These results provide the first molecular evidence for cytoskeletally mediated FP biogenesis.