Roles for two aminopeptidases in vacuolar hemoglobin catabolism in Plasmodium falciparum

During the erythrocytic stage of its life cycle, the human malaria parasite Plasmodium falciparum catabolizes large quantities of host-cell hemoglobin in an acidic organelle, the food vacuole. A current model for the catabolism of globin-derived oligopeptides invokes peptide transport out of the food vacuole followed by hydrolysis to amino acids by cytosolic aminopeptidases. To test this model, we have examined the roles of four parasite aminopeptidases during the erythrocytic cycle. Localization of tagged aminopeptidases, coupled with biochemical analysis of enriched food vacuoles, revealed the presence of amino acid-generating pathways in the food vacuole as well as the cytosol. Based on the localization data and in vitro assays, we propose a specific role for one of the plasmodial enzymes, aminopeptidase P, in the catabolism of proline-containing peptides in both the vacuole and the cytosol. We establish an apparent requirement for three of the four aminopeptidases (including the two food vacuole enzymes) for efficient parasite proliferation. To gain insight into the impact of aminopeptidase inhibition on parasite development, we examined the effect of the presence of amino acids in the culture medium of the parasite on the toxicity of the aminopeptidase inhibitor bestatin. The ability of bestatin to block parasite replication was only slightly affected when 19 of 20 amino acids were withdrawn from the medium, indicating that exogenous amino acids cannot compensate for the loss of aminopeptidase activity. Together, these results support the development of aminopeptidase inhibitors as novel chemotherapeutics directed against malaria.     

Influence of maternal dexamethasone treatment on morphometric characteristics of pituitary GH cells and body weight in near-term rat fetuses

Growth hormone (GH) and glucocorticoids have a powerful influence on controlling fetal growth, differentiation and maturation of numerous tissues. In the present study, the effect of maternal dexamethasone (Dx) treatment on GH cells and body weight in 19- and 21-day-old rat fetuses was investigated using immunocytochemical and morphometric methods. Pregnant female rats received daily injections of 1.0-0.5-0.5 mg Dx/kg b.w. on days 16-18 of pregnancy (experimental group), while the control group received an equal volume of saline. Dx treatment of pregnant rats enhanced immunostaining intensity and significantly increased (p<0.05) GH nuclear and cell volume, as well as volume density and number of GH cells per square millimeter in 19-day-old fetuses compared to the controls. In 21-day-old fetuses after maternal Dx administration, immunoreactivity, volume density and number of GH cells remained significantly increased (p<0.05). Dx treatment of pregnant rats resulted in marked body weight reduction of 21-day-old but not 19 days old fetuses in comparison with the corresponding controls. The presented results demonstrate that maternal Dx application has pronounced effect on morphometric parameters of GH cells of 19- and 21-day-old fetuses. Also, in near-term rat fetuses body weight was largely independent of pituitary GH cell activity.     

ERp57 is essential for efficient folding of glycoproteins sharing common structural domains

ERp57 is a member of the protein disulphide isomerase family of oxidoreductases, which are involved in native disulphide bond formation in the endoplasmic reticulum of mammalian cells. This enzyme has been shown to be associated with both calnexin and calreticulin and, therefore, has been proposed to be a glycoprotein-specific oxidoreductase. Here, we identify endogenous substrates for ERp57 by trapping mixed disulphide intermediates between enzyme and substrate. Our results demonstrate that the substrates for this enzyme are mostly heavily glycosylated, disulphide bonded proteins. In addition, we show that the substrate proteins share common structural domains, indicating that substrate specificity may involve specific structural features as well as the presence of an oligosaccharide side chain. We also show that the folding of two of the endogenous substrates for ERp57 is impaired in ERp57 knockout cells and that prevention of an interaction with calnexin or calreticulin perturbs the folding of some, but not all, substrates with multiple disulphide bonds. These results suggest a specific role for ERp57 in the isomerisation of non-native disulphide bonds in specific glycoprotein substrates.     

Role of 4-hydroxynonenal and its metabolites in signaling

Available evidence from a multitude of studies on the effects of 4-hydroxynonenal (HNE) on cellular processes seem to converge on some common themes: (i) concentration-dependent opposing effects of HNE on key signaling components (e.g. protein kinase C, adenylate cyclase) predict that certain constitutive levels of HNE may be needed for normal cell functions - lowering of this constitutive HNE level in cells promotes proliferative machinery while an increase in this level promotes apoptotic signaling; (ii) HNE is a common denominator in stress-induced apoptosis caused by H(2)O(2), superoxide, UV, heat or oxidant chemicals such as doxorubicin; and (iii) HNE can modulate ligand-independent signaling by membrane receptors such as EGFR or Fas (CD95) and may act as a sensor of external stimuli for eliciting stress-response. Against a backdrop of various reported effects of HNE, in vitro and in vivo, we have critically evaluated the above mentioned hypotheses suggesting a key role of HNE in signaling.     

Obligatory role for phospholipase C-gamma(1) in villin-induced epithelial cell migration

While there is circumstantial evidence to suggest a requirement for phospholipase C-₁ (PLC-₁) in actin reorganization and cell migration, few studies have examined the direct mechanisms that link regulators of the actin cytoskeleton with this crucial signaling molecule. This study was aimed to examine the role that villin, an epithelial cell-specific actin-binding protein, and its ligand PLC-₁ play in migration in intestinal and renal epithelial cell lines that endogenously or ectopically express human villin. Basal as well as epidermal growth factor (EGF)-stimulated cell migration was accompanied by tyrosine phosphorylation of villin and its association with PLC-₁. Inhibition of villin phosphorylation prevented villin-PLC-₁ complex formation as well as villin-induced cell migration. The absolute requirement for PLC-₁ in villin-induced cell migration was demonstrated by measuring cell motility in PLC-₁^–/– cells and by downregulation of endogenous PLC-₁. EGF-stimulated direct interaction of villin with the Src homology domain 2 domain of PLC-₁ at the plasma membrane was demonstrated in living cells by using fluorescence resonance energy transfer. These results demonstrate that villin provides an important link between the activation of phosphoinositide signal transduction pathway and epithelial cell migration. fluorescence resonance energy transfer; actin     

Pulmonary Artery Pseudoaneurysm in COVID-19-Associated Pulmonary Mucormycosis: Case Series and Systematic Review of the Literature

Mycopathologia - Literature on COVID-19-associated pulmonary mucormycosis (CAPM) is sparse. Pulmonary artery pseudoaneurysm (PAP) is an uncommon complication of pulmonary mucormycosis (PM), and...     

A Novel Link between Fic (Filamentation Induced by cAMP)-mediated Adenylylation/AMPylation and the Unfolded Protein Response

The maintenance of endoplasmic reticulum (ER) homeostasis is a critical aspect of determining cell fate and requires a properly functioning unfolded protein response (UPR). We have discovered a previously unknown role of a post-translational modification termed adenylylation/AMPylation in regulating signal transduction events during UPR induction. A family of enzymes, defined by the presence of a Fic (filamentation induced by cAMP) domain, catalyzes this adenylylation reaction. The human genome encodes a single Fic protein, called HYPE (Huntingtin yeast interacting protein E), with adenylyltransferase activity but unknown physiological target(s). Here, we demonstrate that HYPE localizes to the lumen of the endoplasmic reticulum via its hydrophobic N terminus and adenylylates the ER molecular chaperone, BiP, at Ser-365 and Thr-366. BiP functions as a sentinel for protein misfolding and maintains ER homeostasis. We found that adenylylation enhances BiP''s ATPase activity, which is required for refolding misfolded proteins while coping with ER stress. Accordingly, HYPE expression levels increase upon stress. Furthermore, siRNA-mediated knockdown of HYPE prevents the induction of an unfolded protein response. Thus, we identify HYPE as a new UPR regulator and provide the first functional data for Fic-mediated adenylylation in mammalian signaling.     

Expression of a bacterial 3‐dehydroshikimate dehydratase reduces lignin content and improves biomass saccharification efficiency

Lignin confers recalcitrance to plant biomass used as feedstocks in agro‐processing industries or as source of renewable sugars for the production of bioproducts. The metabolic steps for the synthesis of lignin building blocks belong to the shikimate and phenylpropanoid pathways. Genetic engineering efforts to reduce lignin content typically employ gene knockout or gene silencing techniques to constitutively repress one of these metabolic pathways. Recently, new strategies have emerged offering better spatiotemporal control of lignin deposition, including the expression of enzymes that interfere with the normal process for cell wall lignification. In this study, we report that expression of a 3‐dehydroshikimate dehydratase (QsuB from Corynebacterium glutamicum) reduces lignin deposition in Arabidopsis cell walls. QsuB was targeted to the plastids to convert 3‐dehydroshikimate – an intermediate of the shikimate pathway – into protocatechuate. Compared to wild‐type plants, lines expressing QsuB contain higher amounts of protocatechuate, p‐coumarate, p‐coumaraldehyde and p‐coumaryl alcohol, and lower amounts of coniferaldehyde, coniferyl alcohol, sinapaldehyde and sinapyl alcohol. 2D‐NMR spectroscopy and pyrolysis‐gas chromatography/mass spectrometry (pyro‐GC/MS) reveal an increase of p‐hydroxyphenyl units and a reduction of guaiacyl units in the lignin of QsuB lines. Size‐exclusion chromatography indicates a lower degree of lignin polymerization in the transgenic lines. Therefore, our data show that the expression of QsuB primarily affects the lignin biosynthetic pathway. Finally, biomass from these lines exhibits more than a twofold improvement in saccharification efficiency. We conclude that the expression of QsuB in plants, in combination with specific promoters, is a promising gain‐of‐function strategy for spatiotemporal reduction of lignin in plant biomass.     

Arsenic, cadmium and neuron specific enolase (ENO2, γ-enolase) expression in breast cancer

Background

Neuron specific enolase (ENO2, γ-enolase) has been used as a biomarker to help identify neuroendocrine differentiation in breast cancer. The goal of the present study was to determine if ENO2 expression in the breast epithelial cell is influenced by the environmental pollutants, arsenite and cadmium. Acute and chronic exposure of MCF-10A cells to As⁺³ and Cd⁺² sufficient to allow colony formation in soft agar, was used to determine if ENO2 expression was altered by these pollutants.

Results

It was shown that both As⁺³ and Cd⁺² exposure caused significant increases in ENO2 expression under conditions of both acute and chronic exposure. In contrast, ENO1, the major glycolytic enolase in non-muscle and neuronal cells, was largely unaffected by exposure to either As⁺³ or Cd⁺². Localization studies showed that ENO2 in the MCF-10A cells transformed by As⁺³ or Cd⁺² had both a cytoplasmic and nuclear localization. In contrast, ENO1 was localized to the cytoplasm. ENO2 localized to the cytoplasm was found to co-localized with ENO1.

Conclusion

The results are the first to show that ENO2 expression in breast epithelial cells is induced by acute and chronic exposure to As⁺³ or Cd⁺². The findings also suggest a possible link between As⁺³ and Cd⁺² exposure and neuroendocrine differentiation in tumors. Overall, the results suggest that ENO2 might be developed as a biomarker indicating acute and/or chronic environmental exposure of the breast epithelial cell to As⁺³ and Cd⁺².     

Incorporating population-level variation in thermal performance into predictions of geographic range shifts

Determining how species' geographic ranges are governed by current climates and how they will respond to rapid climatic change poses a major biological challenge. Geographic ranges are often spatially fragmented and composed of genetically differentiated populations that are locally adapted to different thermal regimes. Tradeoffs between different aspects of thermal performance, such as between tolerance to high temperature and tolerance to low temperature or between maximal performance and breadth of performance, suggest that the performance of a given population will be a subset of that of the species. Therefore, species-level projections of distribution might overestimate the species' ability to persist at any given location. However, current approaches to modeling distributions often do not consider variation among populations. Here, we estimated genetically-based differences in thermal performance curves for growth among 12 populations of the scarlet monkeyflower, Mimulus cardinalis, a perennial herb of western North America. We inferred the maximum relative growth rate (RGR(max)), temperature optimum (T(opt)), and temperature breadth (T(breadth)) for each population. We used these data to test for tradeoffs in thermal performance, generate mechanistic population-level projections of distribution under current and future climates, and examine how variation in aspects of thermal performance influences forecasts of range shifts. Populations differed significantly in RGR(max) and had variable, but overlapping, estimates of T(opt) and T(breadth). T(opt) declined with latitude and increased with temperature of origin, consistent with tradeoffs between performances at low temperatures versus those at high temperatures. Further, T(breadth) was negatively related to RGR(max), as expected for a specialist-generalist tradeoff. Parameters of the thermal performance curve influenced properties of projected distributions. For both current and future climates, T(opt) was negatively related to latitudinal position, while T(breadth) was positively related to projected range size. The magnitude and direction of range shifts also varied with T(opt) and T(breadth), but sometimes in unexpected ways. For example, the fraction of habitat remaining suitable increased with T(opt) but decreased with T(breadth). Northern limits of all populations were projected to shift north, but the magnitude of shift decreased with T(opt) and increased with T(breadth). Median latitude was projected to shift north for populations with high T(breadth) and low T(opt), but south for populations with low T(breadth) and high T(opt). Distributions inferred by integrating population-level projections did not differ from a species-level projection that ignored variation among populations. However, the species-level approach masked the potential array of divergent responses by populations that might lead to genotypic sorting within the species' range. Thermal performance tradeoffs among populations within the species' range had important, but sometimes counterintuitive, effects on projected responses to climatic change.