Molecular cloning, characterization and expression of PmRsr1, a Ras-related gene from yeast form of Penicillium marneffei

GTP-binding proteins such as Ras act as molecular switches in a large number of signal pathways. In this report, we isolated and characterized a novel Ras small monomeric GTPase Rsr1 gene, designated PmRsr1, from yeast-form Penicillium marneffei. The full-length PmRsr1 cDNA sequence is 1,866 bp in size, and contains an open reading frame of 642 bp encoding 213 amino acids. The predicted molecular mass of PmRsr1 is 24.41 kDa with an estimated theoretical isoelectric point of 9.21. The deduced amino acid sequence of PmRsr1 shows 87% identity with that of Aspergillus fumigatus and A. clavatus. Eight exons and seven introns are identified within the 2,102 bp PmRsr1 genomic DNA sequence of P. marneffei. The open reading frame was subcloned into the pcDNA6-myc-His B expression vector, and the recombinant plasmid was transfected into Vero cell line. The expressed fusion protein was analyzed by SDS-PAGE and western blotting. Differential expression of the PmRsr1 was demonstrated by real-time RT-PCR. The expression of PmRsr1 was the highest in the yeast phase comparing with that in the mycelia and conidia phases.     

Combined Effects of Fluoride and Cadmium on Liver and Kidney Function in Male Rats

Although cadmium (Cd) and fluoride may both have adverse effects on bone, most studies focus on a single agent. In this study, we investigated the effects of cadmium and fluoride on bone at a relative low level. Sprague–Dawley male rats were assigned randomly into four groups which were given sodium chloride, cadmium (50mg/L), and fluoride (20mg/L) alone, or in combination via drinking water. At the 12th week, urine, blood, and bone tissues were collected for biomarker assay, biomechanical assay, and histological assay. Cadmium had significantly adverse effects on bone mineral density, bone biomechanical property, and bone microstructure. Fluoride slightly increased vertebral bone mineral density but negatively affected bone biomechanical property and bone microstructure. Fluoride could reverse the decrease of vertebral bone mineral density caused by cadmium but could not improve the damage of bone biomechanical property and microstructure caused by cadmium. Tartrate-resistant acid phosphatase 5b levels in rats treated with cadmium and fluoride or in combination were 1–2.5 folds higher than the control. Our data suggest that low level of fluoride could reverse the decrease of vertebral bone mineral density caused by cadmium exposure but has no influence on appendicular skeleton damage caused by cadmium.     

Ceramides and sphingosine-1-phosphate mediate the distinct effects of M1/M2-macrophage infusion on liver recovery after hepatectomy

Post-hepatectomy liver dysfunction is a life-threatening morbidity that lacks efficient therapy. Bioactive lipids involved in macrophage polarization crucially regulate tissue injury and regeneration. Herein, we investigate the key bioactive lipids that mediate the cytotherapeutic potential of polarized-macrophage for post-hepatectomy liver dysfunction. Untargeted lipidomics identified elevation of ceramide (CER) metabolites as signature lipid species relevant to M1/M2 polarization in mouse bone-marrow-derived-macrophages (BMDMs). M1 BMDMs expressed a CER-generation-metabolic pattern, leading to elevation of CER; M2 BMDMs expressed a CER-breakdown-metabolic pattern, resulting in upregulation of sphingosine-1-phosphate (S1P). After infusing M1- or M2-polarized BMDMs into the mouse liver after hepatectomy, we found that M1-BMDM infusion increased M1 polarization and CER accumulation, resulting in exaggeration of hepatocyte apoptosis and liver dysfunction. Conversely, M2-BMDM infusion enhanced M2 polarization and S1P generation, leading to alleviation of liver dysfunction with improved hepatocyte proliferation. Treatment of exogenous CER and S1P or inhibition CER and S1P synthesis by siRNA targeting relevant enzymes further revealed that CER induced apoptosis while S1P promoted proliferation in post-hepatectomy primary hepatocytes. In conclusion, CER and S1P are uncovered as critical lipid mediators for M1- and M2-polarized BMDMs to promote injury and regeneration in the liver after hepatectomy, respectively. Notably, the upregulation of hepatic S1P induced by M2-BMDM infusion may have therapeutic potential for post-hepatectomy liver dysfunction.Subject terms: Cell growth, Metabolomics     

Onopordopicrin from the new genus Shangwua as a novel thioredoxin reductase inhibitor to induce oxidative stress-mediated tumor cell apoptosis

Isolation and identification of natural products from plants is an essential approach for discovering drug candidates. Herein we report the characterization of three sesquiterpene lactones from a new genus Shangwua, e.g. onopordopicrin (ONP), C2, and C3, and evaluation of their pharmacological functions in interfering cellular redox signaling. Compared to C2 and C3, ONP shows the most potency in killing cancer cells. Further experiments demonstrate that ONP robustly inhibits thioredoxin reductase (TrxR), which leads to perturbation of cellular redox homeostasis with the favor of oxidative stress. Knockdown of the TrxR sensitizes cells to the ONP treatment while overexpression of the enzyme reduces the potency of ONP, underpinning the correlation of TrxR inhibition to the cytotoxicity of ONP. The discovery of ONP expands the library of the natural TrxR inhibitors, and the disclosure of the action mechanism of ONP provides a foundation for the further development of ONP as an anticancer agent.     

Fluorescent measurement of lipid content in the model organism Chlamydomonas reinhardtii

The green alga Chlamydomonas reinhardtii is one of the most studied microalgae, which has the potential to be used as a model system to study lipid metabolism. Establishment of a method in this organism for rapid and simple measurement of neutral lipids is desirable. Fluorescent measurement of neural lipids by Nile Red staining has been widely used in various cell types including microalgae. However, a systematic study of Nile Red staining to measure neutral lipids in Chlamydomonas has not been reported. Here, we show that Nile Red staining is suitable for relative and absolute quantification of neutral lipids as well as for possible large-scale screening for mutants defective in lipid accumulation. We have compared and optimized the factors involved Nile Red staining including solvents, cell concentration, staining time, and Nile Red concentration. We determined that 5 % DMSO with 1 μg mL^?1 Nile Red and 5–15-min time window after staining was optimal for measuring lipid content of cells within the range of 1 to 8?×?10⁶ cells mL^?1. The absolute quantification of neutral lipids could be achieved by standard addition method. In addition, we developed a protocol that could be potentially used for large-scale screening for cells with different lipid content. Thus, the work reported here provides timely needed techniques to facilitate Chlamydomonas to be used as a model organism for studying lipid metabolism for biodiesel production.     

Development of In Vitro Macrophage System to Evaluate Phagocytosis and Intracellular Fate of Penicillium marneffei Conidia

Penicillium marneffei is a pathogenic fungus that can cause a life-threatening systemic mycosis in the immunocompromised hosts. We established the model for the phagocytosis of P. marneffei conidia by RAW264.7 murine macrophages and designated the fate of P. marneffei in RAW264.7 cells with respect to persistence, phagosome–lysosome-fusion. And we impaired the immune status of mouse and compared the fate and phagosome–lysosome-fusion of P. marneffei in immunocompetent and immunosuppressed mouse peritoneal macrophages cells. We found that conidia could germinate and survive in macrophages. Within 30 min and up to 2 h of heat-killed conidia internalization, the majority of all phagosome types were labeled for the EEA1 (endosomal markers) and LAMP-1 (lysosomal markers), respectively. But both the percentages of LAMP-1 and EEA1 that associated with live conidia were significantly lower than that with heat-killed conidia. Administration of cyclophosphamide resulted in a significant suppression of macrophages function (phagocytic and fungicidal) against P. marneffei that were not apparently seen. Our data provide the evidence that (i) intracellular conversion of P. marneffei conidia into yeast cells still could be observed in macrophages. (ii) Phagosomes containing live Penicillium marneffei conidia might inhibit the phagosome–lysosome-fusion and result to no acidification surrounding the organisms. (iii) Immunity impaired by cyclophosphamide could not influence the function, including phagocytosis, fungicidal activity and phagosome–lysosome-fusion, of macrophages against P. marneffei.     

A NIMA-related kinase,CNK4, regulates ciliary stability and length

NIMA-related kinases (Nrks or Neks) have emerged as key regulators of ciliogenesis. In human, mutations in Nek1 and Nek8 cause cilia-related disorders. The ciliary functions of Nrks are mostly revealed by genetic studies; however, the underlying mechanisms are not well understood. Here we show that a Chlamydomonas Nrk, CNK4, regulates ciliary stability and length. CNK4 is localized to the basal body region and the flagella. The cnk4-null mutant exhibited long flagella, with formation of flagellar bulges. The flagella gradually became curled at the bulge formation site, leading to flagellar loss. Electron microscopy shows that the curled flagella involved curling and degeneration of axonemal microtubules. cnk4 mutation resulted in flagellar increases of IFT trains, as well as its accumulation at the flagellar bulges. IFT speeds were not affected, however, IFT trains frequently stalled, leading to reduced IFT frequencies. These data are consistent with a model in which CNK4 regulates microtubule dynamics and IFT to control flagellar stability and length.     

IFT54 directly interacts with kinesin‐II and IFT dynein to regulate anterograde intraflagellar transport

The intraflagellar transport (IFT) machinery consists of the anterograde motor kinesin‐II, the retrograde motor IFT dynein, and the IFT‐A and ‐B complexes. However, the interaction among IFT motors and IFT complexes during IFT remains elusive. Here, we show that the IFT‐B protein IFT54 interacts with both kinesin‐II and IFT dynein and regulates anterograde IFT. Deletion of residues 342–356 of Chlamydomonas IFT54 resulted in diminished anterograde traffic of IFT and accumulation of IFT motors and complexes in the proximal region of cilia. IFT54 directly interacted with kinesin‐II and this interaction was strengthened for the IFT54^Δ342–356 mutant in vitro and in vivo. The deletion of residues 261–275 of IFT54 reduced ciliary entry and anterograde traffic of IFT dynein with accumulation of IFT complexes near the ciliary tip. IFT54 directly interacted with IFT dynein subunit D1bLIC, and deletion of residues 261–275 reduced this interaction. The interactions between IFT54 and the IFT motors were also observed in mammalian cells. Our data indicate a central role for IFT54 in binding the IFT motors during anterograde IFT.