T cells

We examined the cytotoxic potential of nine N-[2-substituted-2-(2-thienyl) ethyl] piperazinyl quinolone derivatives on human oral epithelial mouth carcinoma (KB) and human squamous carcinoma (A431) cell lines. Phototoxic properties of these compounds were ...

Multiple sclerosis (MS) is a CNS-demyelinating disease characterised by relapsing and chronic neurological impairment. While traditionally CNS autoantigen-specific CD4+ T cells have been considered the culprits in the initial phase of the disease, recent observations have altered this concept. It is now recognised that other T lymphocyte subclasses can initiate CNS demyelination. In addition, other cell types and molecules may play an important role in MS pathogenesis. There is overwhelming evidence that MS is a dynamic process, in which recurrent episodes of blood–brain barrier disruption and CNS inflammation play a crucial role in early disease stages, leading eventually to the largely irreversible changes of demyelination, gliosis and axonal degeneration. These observations may have important therapeutic implications. Within the last ten years, several medications have been approved for MS treatment. These agents, all of which are given parenterally, are only partially effective and are often associated with adverse effects and potential toxicities. The number and different types of medications used for MS are likely to increase in the near future, as several novel therapies are currently tested in clinical trials. 3-hydroxy-3-methyglutaryl coenzyme A reductase inhibitors, ‘statins’, are cholesterol-lowering drugs that are given orally, are safe and have biological effects independent of their cholesterol-reducing properties. Recent reports have shown that statins have anti-inflammatory and neuroprotective properties that may be beneficial in the treatment of MS. This article will outline experimental evidence that suggests potential clinical benefits of statins for MS patients.

The detection of DNA-incorporated bromodeoxyuridine (BrdUrd) in mammalian cells is a well-known and important technique to study cell cycle. The use of TO-PRO-3 for detection of BrdUrd substitution of DNA by dual-laser flow cytometry has been investigated. Fluorescence enhancement of TO-PRO-3 in BrdUrd-labeled cells is registered in combination with the fluorescence emission of the intercalating dye propidium iodide (PI) as a total DNA stain to give bivariate DNA/BrdUrd histograms. By the low concentration of only 0.3 mircoM TO-PRO-3, BrdUrd detection is optimized, and undisturbed total DNA content by PI can be detected as well. TO-PRO-3 is excited by a red HeNe laser and PI by an argon ion laser. In order to understand the binding of TO-PRO-3, energy transfer from PI to TO-PRO-3 has been measured as well as the influence of an external DNA binding dye such as Hoechst 33258 with Adenine-Thymine (AT) binding specificity. Cell cycle studies of human SCL-2 keratinocytes and mouse 3T3 c...

Signal transducers and activators of transcription (STATs) were originally identified as key components of signaling pathways involved in mediating responses to IFNs. Previous studies showed that the Src oncoprotein constitutively activates one STAT family member, Stat3. In this study, we investigated STAT activation in a panel of rodent fibroblast cell lines stably transformed by diverse viral oncoproteins. Using a temperature-sensitive

HUVEC or mouse 3T3 cells infected with SV-40 generate within 3 to 5 days post-infection an ENOX2 species corresponding to the exon-4 minus splice variant of a tumor-associated NADH oxidase (ENOX2 or tNOX) expressed at the cancer cell surface. This study was to seek evidence for splicing factors that might direct formation of the exon 4 minus ENOX2 splice variant. To determine if silencing of ENOX2 exon 4 occurs because of motifs located in exon 4, transfections were performed on MCF-10A (mammary non-cancer), BT-20 (mammary cancer), and HeLa (cervical cancer) cells using a GFP minigene construct containing either a constitutively spliced exon (albumin exon 2) or the alternatively spliced ENOX2 exon 4 between the two GFP halves. Removal of exon 4 from the processed RNA of the GFP minigene construct occurred with HeLa and to a lesser extent with BT-20 but not in non-cancer MCF-10A cells. The Splicing Rainbow Program was used to identify all of the possible hnRNPs binding sites of exon 4 of ENOX2. There are 8 Exonic Splicing Silencers (ESSs) for hnRNP binding in the exon 4 sequences. Each of these sites were mutated by site-directed mutagenesis to test if any were responsible for the splicing skip. Results showed MutG75 ESS mutation changed the GFP expression which is a sign of splicing silence, while other mutations did not. As MutG75 changed the ESS binding site for hnRNP F, this result suggests that hnRNP F directs formation of the exon 4 minus variant of ENOX2.

Odontogenic ameloblast-associated protein (ODAM) has been shown to be specifically expressed in ameloblasts and odontoblasts and has been suggested to play a role in the mineralization of the enamel, possibly through the regulation of matrix metalloproteinase 20. However, its function in dentin is not clear. The purpose of this study was to evaluate the effect of ODAM on tertiary dentin formation. MDPC-23 odontoblastic cells were cultured, and the effect of recombinant ODAM (rODAM) on mineralized nodule formation was evaluated. Pinpoint pulp exposures were made in rat teeth and then capped with rODAM mixed with a carrier (rODAM group), carrier only (Carrier group), or white mineral trioxide aggregate (WMTA group). After 1, 2, and 4 weeks, odontoblasts and tertiary dentin were investigated histologically and immunohistochemically. Nodule formation in MDPC-23 cells was enhanced by rODAM treatment. Odontoblasts were polarized and showed a palisade arrangement in the remaining pulp from...

Adenovirus type 5 E4 open reading frame 4 (E4orf4) protein has been previously shown to counteract transactivation of the junB and c-fos genes by cyclic AMP plus E1A protein and to interact with protein phosphatase 2A (PP2A). Here, we show that the wild-type E4orf4 protein induces apoptosis in the E1A-expressing 293 cells, in NIH 3T3 cells transformed with v-Ras, and in the lung carcinoma cell line H1299. The induction of apoptosis is not accompanied by enhanced levels of p53 in 293 cells and occurs in the absence of p53 in H1299 cells, indicating involvement of a p53-independent pathway. A mutant E4orf4 protein that had lost the ability to induce apoptosis also lost its ability to bind PP2A. We suggest that E4orf4 antagonizes continuous signals to proliferate, like those given by E1A or v-Ras, and that the conflicting signals lead to the induction of cell death.

Since 1997 the National Center for Documentation and Evaluation of Alternative Methods to Animal Experiments, ZEBET, in Berlin, has been coordinating a validation study aimed at prevalidation and validation of three in vitro embryotoxicity tests, funded by the European Center for the Validation of Alternative Methods (ECVAM) at the Joint Research Center (JRC, Ispra, Italy). The tests use the cultivation of postimplantation rat whole embryos (WEC test), cultures of primary limb bud cells of rat embryos (micromass or, MM, test), and cultures of a pluripotent mouse embryonic stem cell line (embryonic stem cell test or EST). Each of the tests was performed in four laboratories under blind conditions. In the preliminary phase of the validation study 6 out of 20 test chemicals comprising different embryotoxic potential (non, weakly, and strongly embryotoxic) were tested. The results were used to define biostatistically based prediction models (PMs) to identify the embryotoxic potential of...

Incidence of type II diabetes is rapidly increasing worldwide. In order to identify complementary or alternative approaches to existing medications, we studied anti-diabetic properties of Vaccinium angustifolium Ait., a natural health product recommended for diabetes treatment in Canada. Ethanol extracts of root, stem, leaf, and fruit were tested at 12.5 μg/ml for anti-diabetic activity in peripheral tissues and pancreatic β cells using a variety of cell-based bioassays. Specifically, we assessed: (1) deoxyglucose uptake in differentiated C2C12 muscle cells and 3T3-L1 adipocytes; (2) glucose-stimulated insulin secretion (GSIS) in β TC-tet pancreatic β cells; (3) β cell proliferation in β TC-tet cells; (4) lipid accumulation in differentiating 3T3-L1 cells; (5) protection against glucose toxicity in PC12 cells. Root, stem, and leaf extracts significantly enhanced glucose transport in C2C12 cells by 15–25% in presence and absence of insulin after 20 h of incubation; no enhancement resulted from a 1 h exposure. In 3T3 cells, only the root and stem extracts enhanced uptake, and this effect was greater after 1 h than after 20 h; uptake was increased by up to 75% in absence of insulin. GSIS was potentiated by a small amount in growth-arrested β TC-tet cells incubated overnight with leaf or stem extract. However, fruit extracts were found to increase 3H-thymidine incorporation in replicating β TC-tet cells by 2.8-fold. Lipid accumulation in differentiating 3T3-L1 cells was accelerated by root, stem, and leaf extracts by as much as 6.5-fold by the end of a 6-day period. Stem, leaf, and fruit extracts reduced apoptosis by 20–33% in PC12 cells exposed to elevated glucose for 96 h. These results demonstrate that V. angustifolium contains active principles with insulin-like and glitazone-like properties, while conferring protection against glucose toxicity. Enhancement of proliferation in β cells may represent another potential anti-diabetic property. Extracts of the Canadian blueberry thus show promise for use as a complementary anti-diabetic therapy.