Ceria-Doped Titania Nanoparticles as Drug Delivery System.

The development of new devices at the nanoscale level, as therapeutic support in medical practice, has facilitated the development of drug delivery systems (DDSs) based on nanoparticles. This enables the transport of an effective dose of therapeutic agents to target cells or tissues, with no collateral damage to healthy cells. Owing to their unique properties, nanoparticles doped with rare earths have demonstrated the potential of being used as a DDS of drug molecules to target cells or tissues. In the present work, ceria-doped titania nanoparticles (CeO2/TiO2NPs) were used to form the DNR-CeO2/TiO2NPs complex as a DDS of daunorubicin (DNR), which was tested in a B-lymphocyte cell culture. The CeO2/TiO2NPs were synthesized via the sol-gel process in a microemulsion of reverse micelle. In general, the results indicated that the nanoparticles presented good biocompatibility and load efficiency superior to that reported in other investigations for pure titania nanoparticles, suggesting that the CeO2/TiO2NPs increased intracellular accumulation of the drug. These results indicate that a delivery strategy using CeO2/TiO2NPs is a promising approach in the medical field, particularly in anticancer therapies.

Performance of culture media for the isolation and identification of Staphylococcus aureus from bovine mastitis.

Rapid isolation and identification of pathogens is a major goal of diagnostic microbiology. In order to isolate and identify Staphylococcus aureus, a number of authors have used a variety of selective and/or differential culture media. However, to date, there are no reports comparing the efficacy of selective and differential culture media for S. aureus isolation from bovine mastitis cases using the 16S rRNA (rrs) gene sequence as a gold standard test. In the present study, we evaluated the efficacy of four selective and/or differential culture media for the isolation of S. aureus from milk samples collected from cows suffering from bovine mastitis. Four hundred and forty isolates were obtained using salt-mannitol agar (SMA, Bioxon), Staphylococcus-110 agar (S110, Bioxon), CHROMAgar Staph aureus (CSA, BD-BBL) and sheep's blood agar (SBA, BD-BBL). All bacterial isolates were identified by their typical colony morphology in the respective media, by secondary tests (for coagulase and ß-haemolysis) and by partial 16S rRNA (rrs) gene sequencing as a gold standard test. Sensitivity, positive predictive and negative predictive values were higher for SMA (86.96, 52.63 and 95.95%, respectively) compared with S110 (70.00, 23.73 and 90.91%, respectively), CSA (69.23, 28.13 and 95.74%, respectively) and SBA (68.75, 37.93 and 89.58%, respectively) while specificity values were similar for all media. Data indicated that the use of culture media for S. aureus isolation combined with determination of coagulase activity and haemolysis as secondary tests improved accuracy of the identification and was in accordance with rrs gene sequence-analysis compared with the use of the culture media alone.

Peroxisome proliferator-activated receptors: role of isoform gamma in the antineoplastic effect of iodine in mammary cancer.

Peroxisome proliferator-activated receptors (PPAR) are ligand-activated transcription factors. Three subtypes--PPAR alpha, PPAR beta, and PPAR gamma--have been identified and are differentially expressed in tissues. Originally, they were described as molecular regulators of lipid metabolism; recently, it has been shown that they are also involved in regulating the cell cycle and apoptosis in both normal and tumoral cells. In fact, some synthetic PPAR ligands are used to treat dyslipidemia, metabolic diseases, and type 2 diabetes. Here, we review the role of PPAR gamma (PPARγ) in tumor initiation and progression, emphasizing the relationship between this isoform and the cellular and molecular mechanisms involved in the antineoplastic effect of iodine on mammary cancer.

A complex between 6-iodolactone and the peroxisome proliferator-activated receptor type gamma may mediate the antineoplastic effect of iodine in mammary cancer.

Recently we and other groups have shown that molecular iodine (I(2)) exhibits potent antiproliferative and apoptotic effects in mammary cancer models. In the human breast cancer cell line MCF-7, I(2) treatment generates iodine-containing lipids similar to 6-iodo-5-hydroxy-eicosatrienoic acid and the 6-iodolactone (6-IL) derivative of arachidonic acid (AA), and it significantly decreases cellular proliferation and induces caspase-dependent apoptosis. Several studies have shown that AA is a natural ligand of the peroxisome proliferator-activated receptors (PPARs), which are nuclear transcription factors thought to participate in regulating cancer cell proliferation. Our results show that in MCF-7 cells: (1) 6-IL binds specifically and with high affinity to PPAR proteins (EMSA assays), (2) 6-IL activates both transfected (by transactivation assays) and endogenous (by lipid accumulation) peroxisome proliferator response elements, and (3) 6-IL supplementation increases PPAR gamma and decreases PPAR alpha expression. These results implicate PPARs in a molecular mechanism by which I(2), through formation of 6-IL, inhibits the growth of human breast cancer cells.

Microbial fructosyltransferases and the role of fructans.

Microbial fructosyltransferases are polymerases that are involved in microbial fructan (levan, inulin and fructo-oligosaccharide) biosynthesis. Structurally, microbial fructosyltransferase proteins share the catalytic domain of glycoside hydrolases 68 family and are grouped in seven phylogenetically related clusters. Fructosyltransferase-encoding genes are organized in operons or in clusters associated with other genes related to carbohydrate metabolism or fructosyltransferase secretion. Fructosyltransferase gene expression is mainly regulated by two-component systems or phosphorelay mechanisms that respond to sucrose availability or other environmental signals. Microbial fructans are involved in conferring resistance to environmental stress such as water deprivation, nutrient assimilation, biofilm formation, and as virulence factors in colonization. As a result of the biological and industrial importance of fructans, fructosyltransferases have been the subject of extensive research, conducted to improve their enzymatic activity or to elucidate their biological role in nature.

Internalization of Staphylococcus aureus by bovine endothelial cells is associated with the activity state of NF-kappaB and modulated by the pro-inflammatory cytokines TNF-alpha and IL-1beta.

Bacterial internalization is an important process in the pathogenesis of infectious diseases in which nuclear factor kappaB (NF-kappaB) plays a prominent role. We present pharmacological evidence indicating that in bovine endothelial cells (BEC) the internalization of Staphylococcus aureus, a pathogenic bacterium that causes mastitis in bovine cattle, was associated with the activation of NF-kappaB. The internalization of S. aureus increased when BEC were stimulated with alpha-tumour necrosis factor (TNF-alpha) or beta-interleukin 1 (IL-1beta) which are known activators of NF-kappaB. SN50 (an inhibitor peptide of NF-kappaB nuclear translocation) and BAY 11-7083 (a chemical that inhibits the IkappaBalpha phosphorylation) caused significant reduction in S. aureus intracellular number, indicating that its internalization was associated with the NF-kappaB activity. Furthermore, specific inhibition of c-Jun N-terminal kinase with SP600125 (SP) or p-38 with SB203580 (SB) did not cause any change in the S. aureus intracellular number compared with the untreated control. Finally, TNF-alpha treatment of BEC after the addition of both SP and SB, induced a significant increase in S. aureus internalization above the control value. These data indicate that NF-kappaB activity is associated with S. aureus internalization and suggest that this transcription factor may play a role in the pathophysiology of bovine mastitis caused by this bacterium.

Cloning, structural organization and tissue-specific expression of the rabbit transferrin gene.

We cloned the rabbit transferrin (rTf) cDNA and gene, and quantified the expression of the rTf gene at the RNA level in various organs. The tissue-specific pattern of expression of rTf gene is different to those in other species, with a high expression in mammary gland and kidney. The exon/intron structure of the rTf gene (17 exons/16 introns) is similar to those of transferrins from other species. The sequence of the rTf cDNA already published is corrected and lengthened in the 5' region, and a likely polymorphism is documented.

The RU5 ('R') region from human leukaemia viruses (HTLV-1) contains an internal ribosome entry site (IRES)-like sequence.

RNA fragments containing the complete R region and the beginning of the U5 region ('R') from the human T cell leukaemia virus 1 (HTLV-1) stimulated the translation of the second cistrons in bicistronic mRNAs. The 5' untranslated region from SV40 early genes (SU) which was unable to stimulate translation of second cistrons amplified markedly the internal ribosome entry site (IRES) effect of the HTLV-1 'R' fragments. The 'R' regions from HTLV-1 have therefore properties similar to internal ribosome entry sites (IRES) originally found in picornavirus. The beginning of the U5 region from HTLV-1 contains a polypyrimidine sequence which is known to play an essential role in the IRES activity in picornavirus. The same experiments carried out using the 'R' region from bovine leukaemia virus (BLV) showed that this sequence has at most a weak IRES effect. One retroviruses, HTLV-1 and perhaps others contain therefore an IRES activity. Interestingly, the combined SU 'R' sequence worked efficiently with different cistrons, different promoters and in all tested cell lines, whereas the poliovirus IRES was active in CHO cells but not in the mouse mammary cell line HC11. The SU 'R' sequence may therefore preferably be used to generate active bicistronic mRNAs.

Cell proteins bind to a linear polypyrimidine-rich sequence within the 5'-untranslated region of rhinovirus 14 RNA.

Members of the picornavirus family initiate translation of their RNA genomes by a cap-independent mechanism in which ribosomes bind to an internal site in the 5' untranslated region (5'-UTR). This unique process requires an internal ribosome entry site (IRES), a highly structured RNA whose function is mediated in part by interactions with cell proteins. The IRES element of human rhinovirus 2 (HRV-2) extends from nucleotide (nt) 10 to between nt 544 and 568 and has been shown to interact with two cell proteins, pyrimidine tract-binding protein (pPTB) and p97. To map the specific regions of HRV-14 RNA that bind cell proteins, mobility shift, UV cross-linking and Western immunoblot analyses were performed. The results indicate that an RNA sequence from nt 538 to 591 interacts with pPTB and La, two proteins previously shown to functionally interact with the IRES elements of several picornaviruses. Two additional proteins, p97 and p68, were also cross-linked to nt 538 to 591 RNA. These four proteins interact with a putatively unstructured portion of the 5'-UTR that contains a polypyrimidine tract and has been shown to be present at the 3' border of sequences that are essential for IRES function of HRV-2. These protein-RNA interactions are likely to play a role in internal initiation of translation.

A simple method of DNA extraction from whole tissues and blood using glass powder for detection of transgenic animals by PCR.

A very simple and reliable method to extract DNA directly from mouse tail, rabbit ear and blood is described. Tissue was homogenized in a solution of NaI and the DNA was extracted using glass powder. The extracted DNA was obtained in sufficient quantity and purity to allow direct detection of transgene by PCR.

The effect of matrix attached regions (MAR) and specialized chromatin structure (SCS) on the expression of gene constructs in cultured cells and in transgenic mice.

The flanking sequences of several genes have been shown to direct a position independent expression of transgenes. Attempts to completely identify the insulating sequences have failed so far. Some of these sequences contain a matrix attached region (MAR) located in the flanking part of the genes. This article will show that the MARs in cultured cells located in the 3' OH region of the human apolipoprotein B100 (Apo B100) and within the SV40 genome were unable to stimulate and insultate transgene expression directed by the promoters from a rabbit whey acidic protein (WAP) gene or from human cytomegalovirus (hCMV) early genes. In transgenic mice, the MAR from the Apo B100 and SV40 genes did not enhance the expression of a transgene containing the rabbit whey acid protein (WAP) promotor, the late gene SV40 intron (VP1 intron), the bovine growth hormone (bGH) cDNA and the SV40 late gene terminator. This construct was even toxic for embryos. Similarly, the specialized chromatin structure (SCS) from the Drosophila 87A7 HSP70 gene reduced chloramphenicol acetyl transferase (CAT) activity when added between a cytomegalovirus (CMV) enhancer and a Herpes simplex thymidine kinase (TK) gene promoter. This inhibitory action was almost complete when a second SCS sequence was added before the CMV enhancer. Sequences from the firefly luciferase and from the human gene cathepsin D cDNA used as control unexpectedly showed a similar inhibitory effect when added to the CMVTKCAT construct instead of SCS. When added before the CMV enhancer and after the transcription terminator in the CMVTKCAT construct, the SCS sequence was unable to insulate the integrated gene as seen by the fact that the level of CAT in cell extracts were by no means correlated with the number of copies in individual clones. From these data, it is concluded that i) a MAR containing the canonical AT rich sequences does not amplify the expression of all gene constructs ii) At rich MAR sequences do not have per se an insulating effect iii) Drosophila SCS from the 87A7 HSP70 gene has no insulating effect in all gene constructs (at least in mammalian cells) iv) and the addition of a DNA fragment between an enhancer and a promoter in a gene construct cannot be used as a reliable test to evaluate its insulating property.