Atg28, a novel coiled-coil protein involved in autophagic degradation of peroxisomes in the methylotrophic yeast Pichia pastoris

In methylotrophic yeasts, peroxisomes are required for methanol utilization, but are dispensable for growth on most other carbon sources. Upon adaptation of cells grown on methanol to glucose or ethanol, redundant peroxisomes are selectively and quickly shipped to, and degraded in, vacuoles via a process termed pexophagy. We identified a novel gene named ATG28 (autophagy-related genes) involved in pexophagy in the yeast Pichia pastoris. This yeast exhibits two morphologically distinct pexophagy pathways, micro- and macropexophagy, induced by glucose or ethanol, respectively. Deficiency in ATG28 impairs both pexophagic mechanisms but not general (bulk turnover) autophagy, a degradation pathway in yeast triggered by nitrogen starvation. It is known that the micro-, macropexophagy, and general autophagy machineries are distinct but share some molecular components. The identification of ATG28 suggests that pexophagy may involve species-specific components, since this gene appears to have only weak homologues in other yeasts.     

Function and redox state of mitochondrial localized cysteine-rich proteins important in the assembly of cytochrome c oxidase

The cytochrome c oxidase (CcO) complex of the mitochondrial respiratory chain exists within the mitochondrial inner membrane (IM). The biogenesis of the complex is a multi-faceted process requiring multiple assembly factors that function on both faces of the IM. Formation of the two copper centers of CcO occurs within the intermembrane space (IMS) and is dependent on assembly factors with critical cysteinyl thiolates. Two classes of assembly factors exist, one group being soluble IMS proteins and the second class being proteins tethered to the IM. A common motif in the soluble assembly factors is a duplicated Cx(9)C sequence motif. Since mitochondrial respiration is a major source of reactive oxygen species, control of the redox state of mitochondrial proteins is an important process. This review documents the role of these cysteinyl CcO assembly factors within the IMS and the necessity of redox control in their function.     

Sodium nitroprusside induces mild oxidative stress in Saccharomyces cerevisiae

Nitric oxide is known to be a messenger in animals and plants. It may act either as a pro-oxidant or antioxidant. In the present work, the yeast Saccharomyces cerevisiae was treated under aerobic conditions with the nitric oxide donor, sodium nitroprusside (SNP), at concentrations of 1, 5 and 10 mM. The activities of antioxidant enzymes as well as concentrations of protein carbonyls and cellular thiols were measured. Yeast incubation with SNP increased the activities of catalase and superoxide dismutase. Cycloheximide, an inhibitor of translation, blocked SNP-induced catalase activation, but not SOD activation. Incubation with SNP increased the activity of peroxisomal catalase, whereas cytosolic catalase was not affected. SNP treatment inactivated aconitase in a dose-dependent manner. Surprisingly, in cells incubated with 1 mM SNP, the levels of low-molecular weight thiols were significantly higher, whereas the concentrations of protein carbonyl groups were lower than those in untreated cells. The incubation of yeast cells either with decomposed SNP or with SNP under anaerobic conditions did not result in SOD and catalase activation. It is suggested, that under aerobic conditions, the SNP effects are connected with induction of mild oxidative/nitrosative stress.     

2,4‐DINITROPHENOL PARTIALLY ALLEVIATES FERROCYANIDE‐INDUCED TOXICITY IN Drosophila melanogaster

The toxicity of potassium ferrocyanide (PFC) and protective effects of 2,4‐dinitrophenol (DNP) under PFC treatment were tested on the Drosophila melanogaster model system. Fly larvae were raised on food supplemented with PFC at concentrations of 1.0 mM and mixtures with DNP in concentrations of 0.50 and 1.25 mM, either alone or in combination with 1.0 mM PFC. Food supplementation with PFC decreased larvae viability or pupation height, whereas when larvae were fed by PFC and DNP combination the decrease was less pronounced. Larval exposure to PFC and mixtures of DNP and PFC lowered activities of aconitase. Larval treatment with PFC resulted in higher carbonyl protein, uric acid, and low molecular mass thiols content and higher activity of thioredoxin reductase in adult flies, while DNP in mixtures with PFC relieved these effects. Furthermore, treatment with PFC/DNP mixtures resulted in higher activities of superoxide dismutase and glutathione‐S‐transferase. It is proposed that PFC toxicity is mainly related to the cyanide and iron ions, released during its decomposition. The potential mechanisms of protective DNP effects against PFC toxicity are discussed.     

Stable overproducer of hepatitis B surface antigen in the methylotrophic yeast Hansenula polymorpha due to multiple integration of heterologous auxotrophic selective markers and defect in peroxisome biogenesis

Two methods of multicopy integrant selection in the methylotrophic yeast Hansenula polymorpha based on the use of heterologous yeast auxotrophic genes have been used to isolate effective overproducers of hepatitis B surface antigen (HBsAg). One selection marker was described earlier for this yeast, the Saccharomyces cerevisiae URA3 gene, whereas the second selection marker was developed by us, the Pichia pastoris ADE1 gene with shortened native promoter. Sequential use of both selection markers produced stable transformants containing up to 30 integration cassettes with HBsAg gene. Deletion of PEX3 gene coding for peroxine involved in the early step of peroxisome formation substantially increased the production of HBsAg in glucose medium as compared to the parental strain. Maximal production of HBsAg in Δpex3 strain was nearly 8–9 % of the total cell protein.     

Acute cholesterol-induced anti-natriuretic effects: role of epithelial Na+ channel activity, protein levels, and processing

The epithelial Na(+) channel (ENaC) is modulated by membrane lipid composition. However, the effect of an in vivo change of membrane composition is unknown. We examined the effect of a 70-day enhanced cholesterol diet (ECD) on ENaC and renal Na(+) handling. Rats were fed a standard chow or one supplemented with 1% cholesterol and 0.5% cholic acid (ECD). ECD animals exhibited marked anti-diuresis and anti-natriuresis (40 and 47%), which peaked at 1-3 weeks. Secondary compensation returned urine output and urinary Na(+) excretion to control levels by week 10. During these initial changes, there were no accompanying effects on systolic blood pressure, serum creatinine, or urinary creatinine excretion, indicating that the these effects of ECD preceded those which modify renal filtration and blood pressure. The effects of ECD on ENaC were evaluated by measuring the relative protein content of α, β, and γ subunits. α and γ blots were further examined for subunit cleavage (a process that activates ENaC). No significant changes were observed in α and β levels throughout the study. However, levels of cleaved γ were elevated, suggesting that ENaC was activated. The changes of γ persisted at week 10 and were accompanied by additional subunit fragments, indicating potential changes of γ-cleaving proteases. Enhanced protease activity, and specifically that which could act on the second identified cleavage site in γ, was verified in a newly developed urinary protease assay. These results predict enhanced ENaC activity, an effect that was confirmed in patch clamp experiments of principal cells of split open collecting ducts, where ENaC open probability was increased by 40% in the ECD group. These data demonstrate a complex series of events and a new regulatory paradigm that is initiated by ECD prior to the onset of elevated blood pressure. These events lead to changes of renal Na(+) handling, which occur in part by effects on extracellular γ-ENaC cleavage.     

Endocannabinoids and Inflammatory Response in Periodontal Ligament Cells

Endocannabinoids are associated with multiple regulatory functions in several tissues. The main endocannabinoids, anandamide (AEA) and 2-arachidonylglycerol (2-AG), have been detected in the gingival crevicular fluid of periodontitis patients, but the association between periodontal disease or human periodontal ligament cells (hPdLCs) and endocannabinoids still remain unclear. The aim of the present study was to examine the effects of AEA and 2-AG on the proliferation/viability and cytokine/chemokine production of hPdLCs in the presence/absence of Porphyromonas gingivalis lipopolysaccharide (P. gingivalis LPS). The proliferation/viability of hPdLCs was measured using 3,4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT)-assay. Interleukin-6 (IL-6), interleukin-8 (IL-8), and monocyte chemotactic protein-1 (MCP-1) levels were examined at gene expression and protein level by real-time PCR and ELISA, respectively. AEA and 2-AG did not reveal any significant effects on proliferation/viability of hPdLCs in the absence of P. gingivalis LPS. However, hPdLCs viability was significantly increased by 10–20 µM AEA in the presence of P. gingivalis LPS (1 µg/ml). In the absence of P. gingivalis LPS, AEA and 2-AG did not exhibit any significant effect on the expression of IL-8 and MCP-1 expression in hPdLCs, whereas IL-6 expression was slightly enhanced by 10 µM 2-AG and not affected by AEA. In P.gingivalis LPS stimulated hPdLCs, 10 µM AEA down-regulated gene-expression and protein production of IL-6, IL-8, and MCP-1. In contrast, 10 µM 2-AG had an opposite effect and induced a significant up-regulation of gene and protein expression of IL-6 and IL-8 (P<0.05) as well as gene-expression of MCP-1 in P. gingivalis LPS stimulated hPdLCs. Our data suggest that AEA appears to have an anti-inflammatory and immune suppressive effect on hPdLCs’ host response to P.gingivalis LPS, whereas 2-AG appears to promote detrimental inflammatory processes. In conclusion, AEA and 2-AG might play an important role in the modulation of periodontal inflammation.