Much interest has recently arisen in methods for DNA fingerprinting based on the polymerase chain reaction (PCR).

We report here the expression and properties of the intermediate-conductance Ca²⁺-activated K⁺ (IK_Ca) channel in the GL-15 human glioblastoma cell line. Macroscopic IK_Ca currents on GL-15 cells displayed a mean amplitude of 7.2±0.8 pA/pF at 0 mV, at day 1 after plating. The current was inhibited by clotrimazole (CTL, IC₅₀=257 nM), TRAM-34 (IC₅₀=55 nM), and charybdotoxin (CTX, IC₅₀=10.3 nM). RT-PCR analysis demonstrated the expression of mRNA encoding the IK_Ca channel in GL-15 cells. Unitary currents recorded using the inside-out configuration had a conductance of 25 pS, a K_D for Ca²⁺ of 188 nM at -100 mV, and no voltage dependence. We tested whether the IKCa channel expression in GL-15 cells could be the result of an increased ERK activity. Inhibition of the ERK pathway with the MEK antagonist PD98059 (25 µM, for 5 days) virtually suppressed the IK_Ca current in GL-15 cells. PD98059 treatment also increased the length of cellular processes and up-regulated the astrocytic differentiative marker GFAP. A significant reduction of the IKCa current amplitude was also observed with time in culture, with mean currents of 7.17±0.75 pA/pF at 1-2 days, and 3.11±1.35 pA/pF at 5-6 days after plating. This time-dependent downregulation of the IK_Ca current was not accompanied by changes in the ERK activity, as assessed by immunoblot analysis. Semiquantitative RT-PCR analysis demonstrated a ~35% reduction of the IK_Ca channel mRNA resulting from ERK inhibition and a ~50% reduction with time in culture.

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Nanotechnology exerts an increasing impact on the development of more effective tools for the diagnosis and treatment of human diseases. This applies in particular to central nervous system (CNS) disorders. Development of therapeutics for CNS is, in fact, one of the most challenging areas in drug development, mainly due to the presence of the blood-brain barrier (BBB) which separates the blood from the cerebral parenchyma thus limiting the brain uptake of the vast majority of neurotherapeutic agents. Among the several strategies which have been developed over the last years in order to overcome this problem, nanotechnology-based approaches have gained increasing attention as the most promising strategies for CNS targeted drug delivery. Nanocarriers offer several advantages such as the possibility to maintain drug levels in a therapeutically desirable range, as well as the increase of half-lives, solubility, stability and permeability of drugs. Furthermore, the system can be designed in such a way as to release the drug in a controlled way or in a triggered way. This review focuses on lipid-based nanocarriers and more specifically on liposomes, lipid-core micelles, and lipid nanocapsules, and provides an update on their composition and use, including recent patents in the field.

In order to obtain steroid-independent probes for human progesterone receptor (PR), the A [88-93 kilodalton (kDa)] and B (109-119 kDa) forms of PR from T47D human breast cancer cells were partially purified and used to generate a series of 14 monoclonal antibodies. Initially, unoccupied PR was isolated from cytosol extracts by steroid affinity chromatography, followed by chromatography on diethylaminoethyl Bio-Gel. The partially pure (3-15%) PR consisted of two steroid-binding components that migrated at 89 kDa and 109 kDa in reducing sodium dodecyl sulfate gels after being photoaffinity labeled with the synthetic progestin [3H]R5020. Two unique monoclonal antibodies to PR were derived from a male Lewis rat immunized with this material. One of these antibodies (JU601) was coupled to Sepharose 4B and used to purify T47D nuclear PR for additional immunizations. Highly purified (30-70%) PR migrated as 93 kDa and 119 kDa progestin-binding proteins in sodium dodecyl sulfate gels. In all, thirteen monoclonal antibodies were obtained that recognized epitopes shared by both receptor forms. One mouse immunoglobulin G (KC146) was completely specific for the larger B form. Interestingly, the epitope for this antibody was present on all PRs tested, including the B form of PR from chicken oviduct, whereas nine other antibodies recognized only human PR and the remaining four cross reacted with rabbit PR. With the exception of the JU145 and JU601 rat immunoglobulin Ms, all antibodies appeared to be completely specific for the A or B forms of PR. Each recognized the cytosol and nuclear forms of occupied as well as unoccupied PR. Although the relationship between B and A was not established, it is clear that an amino-terminal region of B is not present in A, and that a significant portion of A and B are either identical or very similar in amino acid sequence.