A Novel Folate-Targeted Nanoliposomal System of Doxorubicin for Cancer Targeting

Targeted drug delivery systems for cancer improves anti-tumor efficacy and reduces systemic toxicity by restricting availability of cytotoxic drugs within tumors. Targeting moieties, such as natural ligands (folic acid, transferrin, and biotin) which are overexpressed on tumors, have been used to enhance liposome-encapsulated drug accumulation within tumors and resulted in better control. In this report, we explored the scope of targeting ligand folic acid, which is incorporated in liposome systems using folic acid-modified cholesterol (CPF), enabled highly selective tumor-targeted delivery of liposome-encapsulated doxorubicin and resulted in increased cytotoxicity within tumors. Folate-tagged poloxamer-coated liposomes (FDL) were found to have significantly higher cellular uptake than conventional poloxamer-coated liposomes (DL), as confirmed by fluorometric analysis in B16F10 melanoma cells. Biodistribution study of the radiolabeled liposomal system indicated the significantly higher tumor uptake of FDL as compared to DL. Anti-tumor activity of FDL against murine B16F10 melanoma tumor-bearing mice revealed that FDL inhibited tumor growth more efficiently than the DL. Taken together, the results demonstrated the significant potential of the folate-conjugated nanoliposomal system for drug delivery to tumors.     

Heterologous production and characterisation of two distinct dihaem-containing membrane integral cytochrome b561 enzymes from Arabidopsis thaliana in Pichia pastoris and Escherichia coli cells

Cytochrome (cyt) b₅₆₁ proteins are dihaem-containing membrane proteins, belonging to the CYBASC (cytochrome-b₅₆₁-ascorbate-reducible) family, and are proposed to be involved in ascorbate recycling and/or the facilitation of iron absorption. Here, we present the heterologous production of two cyt b₅₆₁ paralogs from Arabidopsis thaliana (Acytb₅₆₁-A, Acytb₅₆₁-B) in Escherichia coli and Pichia pastoris, their purification, and initial characterisation. Spectra indicated that Acytb₅₆₁-A resembles the best characterised member of the CYBASC family, the cytochrome b₅₆₁ from adrenomedullary chromaffin vesicles, and that Acytb₅₆₁-B is atypical compared to other CYBASC proteins. Haem oxidation–reduction midpoint potential (E_M) values were found to be fully consistent with ascorbate oxidation activities and Fe^3 +-chelates reductase activities. The ascorbate dependent reduction and protein stability of both paralogs were found to be sensitive to alkaline pH values as reported for the cytochrome b₅₆₁ from chromaffin vesicles. For both paralogs, ascorbate-dependent reduction was inhibited and the low-potential haem E_M values were affected significantly by incubation with diethyl pyrocarbonate (DEPC) in the absence of ascorbate. Modification with DEPC in the presence of ascorbate left the haem E_M values unaltered compared to the unmodified proteins. However, ascorbate reduction was inhibited. We concluded that the ascorbate-binding site is located near the low-potential haem with the Fe^3 +-chelates reduction-site close to the high-potential haem. Furthermore, inhibition of ascorbate oxidation by DEPC treatment occurs not only by lowering the haem E_M values but also by an additional modification affecting ascorbate binding and/or electron transfer. Analytical gel filtration experiments suggest that both cyt b₅₆₁ paralogs exist as homodimers.     

Cilia and Hedgehog: When and how was their marriage solemnized?

Primary cilia are essential for Hedgehog (Hh) signaling in mammals, and this requirement appears to be conserved in other vertebrates as well. Here, I review recent work that has scrutinized the evolution of the link between the Hh pathway and cilia, discuss what we have learnt from these studies and speculate on how this fascinating problem can be further explored.     

Stimulation of toll-like receptor 2 by Coxiella burnetii is required for macrophage production of pro-inflammatory cytokines and resistance to infection

Innate and adaptive immune responses are initiated upon recognition of microbial molecules by Toll-like receptors (TLRs). We have investigated the importance of these receptors in the induction of pro-inflammatory cytokines and macrophage resistance to infection with Coxiella burnetii, an obligate intracellular bacterium and the etiological agent of Q fever. By using a Chinese hamster ovary/CD14 cell line expressing either functional TLR2 or TLR4, we determined that C. burnetii phase II activates TLR2 but not TLR4. Macrophages deficient for TLR2, but not TLR4, produced less tumor necrosis factor-alpha and interleukin-12 upon C. burnetii infection. Furthermore, it was found that TLR2 activation interfered with C. burnetii intracellular replication, as macrophages from TLR2-deficient mice were highly permissive for C. burnetii growth compared with macrophages from wild type mice or TLR4-deficient mice. Although LPS modifications distinguish virulent C. burnetii phase I bacteria from avirulent phase II organisms, electrospray ionization-mass spectrometry analysis showed that the lipid A moieties isolated from these two phase variants are identical. Purified lipid A derived from either phase I or phase II LPS failed to activate TLR2 and TLR4. Indeed, the lipid A molecules were able to interfere with TLR4 signaling in response to purified Escherichia coli LPS. These studies indicate that TLR2 is an important host determinant that mediates recognition of C. burnetii and a response that limits growth of this intracellular pathogen.     

Na+-coupled Cl− transport in the gastric mucosa of the guinea pig

The Cl^–/HCO₃^– exchange mechanism usually postulated to occur in gastric mucosa cannot account for the Na⁺-dependent electrogenic serosal to mucosal Cl^– transport often observed. It was recently suggested that an additional Cl^– transport mechanism driven by the Na⁺ electrochemical potential gradient may be present on the serosal side of the tissue. To verify this, we have studied Cl^– transport in guinea pig gastric mucosa. Inhibiting the (Na⁺, K⁺) ATPase either by serosal addition of ouabain or by establishing K⁺-free mucosal and serosal conditions abolished net Cl^– transport. Depolarizing the cell membrane potential with triphenylmethylphosphonium (a lipid-soluble cation), and hence reducing both the Na⁺ and Cl^– electrochemical potential gradients, resulted in inhibition of net Cl^– flux. Reduction of short-circuit current on replacing Na⁺ by choline in the serosal bathing solution was shown to be due to inhibition of Cl^– transport. Serosal addition of diisothiocyanodisulfonic acid stilbene (an inhibitor of anion transport systems) abolished net Cl^– flux but not net Na⁺ flux. These results are compatible with the proposed model of a Cl^–/Na⁺ cotransport mechanism governing serosal Cl^– entry into the secreting cells. We suggest that the same mechanism may well facilitate both coupled Cl^–/Na⁺ entry and coupled HCO₃^–/Na⁺ exit on the serosal side of the tissue.     

Purification and properties of a carboxymethylcellulase from phytopathogenic fungus Macrophomina phaseolina

From the culture filtrate of Macrophomina phaseolina, two forms of carboxymethylcellulase were separated by ion-exchange chromatography and designated as CMCase-I and CMCase-II. CMCase-I was purified following a four-step procedure involving gel filtration on Sephadex G-75, Con-A Sepharose 4B affinity chromatography, fast protein liquid chromatography on mono Q anion-exchanger and on Superose 12 gel filtration. The final preparation was homogeneous by SDS-PAGE, isoelectric focussing in thin layers of polyacrylamide gels and immunoelectrophoresis. The enzyme showed optimum activity at pH 5.5 and 65 degrees C, was stable to heating at 65 degrees C for 10 min, and retained 31% of original activity after heating at 80 degrees C for 10 min. The molecular weight of the enzyme was 3.5 x 10(4) Da. A Km of 0.25 mg/ml was determined using carboxymethyl-cellulose as the substrate.     

QTL detection for coccidiosis (Eimeria tenella) resistance in a Fayoumi?×?Leghorn F2 cross,using a medium-density SNP panel

Background

Coccidiosis is a major parasitic disease that causes huge economic losses to the poultry industry. Its pathogenicity leads to depression of body weight gain, lesions and, in the most serious cases, death in affected animals. Genetic variability for resistance to coccidiosis in the chicken has been demonstrated and if this natural resistance could be exploited, it would reduce the costs of the disease. Previously, a design to characterize the genetic regulation of Eimeria tenella resistance was set up in a Fayoumi × Leghorn F₂ cross. The 860 F₂ animals of this design were phenotyped for weight gain, plasma coloration, hematocrit level, intestinal lesion score and body temperature. In the work reported here, the 860 animals were genotyped for a panel of 1393 (157 microsatellites and 1236 single nucleotide polymorphism (SNP) markers that cover the sequenced genome (i.e. the 28 first autosomes and the Z chromosome). In addition, with the aim of finding an index capable of explaining a large amount of the variance associated with resistance to coccidiosis, a composite factor was derived by combining the variables of all these traits in a single variable. QTL detection was performed by linkage analysis using GridQTL and QTLMap. Single and multi-QTL models were applied.

Results

Thirty-one QTL were identified i.e. 27 with the single-QTL model and four with the multi-QTL model and the average confidence interval was 5.9 cM. Only a few QTL were common with the previous study that used the same design but focused on the 260 more extreme animals that were genotyped with the 157 microsatellites only. Major differences were also found between results obtained with QTLMap and GridQTL.

Conclusions

The medium-density SNP panel made it possible to genotype new regions of the chicken genome (including micro-chromosomes) that were involved in the genetic control of the traits investigated. This study also highlights the strong variations in QTL detection between different models and marker densities.