Determination of Brain Interstitial Concentrations by Microdialysis

Microdialysis is an extensively used technique for the study of solutes in brain interstitial space. The method is based on collection of substances by diffusion across a dialysis membrane positioned in the brain. The outflow concentration reflects the interstitial concentration of the substance of interest, but the relationship between these two entities is at present unclear. So far, most evaluations have been based solely on calibrations in saline. This procedure is misleading, because the ease by which molecules in saline diffuse into the probe is different from that of tissue. We describe here a mathematical analysis of mass transport into the dialysis probe in tissue based on diffusion equations in complex media. The main finding is that diffusion characteristics of a given substance have to be included in the formula. These include the tortuosity factor (lambda) and the extracellular volume fraction (alpha). We have substantiated this by studies in a well-defined complex medium (red blood cell suspensions) as well as in brain. We conclude that the traditional calculation procedure results in interstitial concentrations that are too low by a factor of lambda 2/alpha for a given compound.     

Sequence-targeted cleavage of single- and double-stranded DNA by oligothymidylates covalently linked to 1,10-phenanthroline

The nuclease activity of 1,10-phenanthroline copper ion was targeted to a specific sequence by attachment of the ligand to the 5' or 3' end of octathymidylates. An acridine derivative was also attached to the other end of the oligothymidylate-phenanthroline conjugate. The duplex formed by the oligothymidylate with its complementary sequence was stabilized by intercalation of the acridine derivative. The reaction induced by 3-mercaptopropionic acid led to a very localized cleavage of a 27-nucleotide-long DNA fragment containing a (dA)8 sequence. At high NaCl concentration or in the presence of spermine, cleavage of the single-stranded 27-mer fragment occurred on both sides of the target sequence. This was ascribed to the formation of a triple helix involving two 1,10-phenanthroline-octathymidylate strands that adopt an antiparallel orientation with respect to each other. When a 27-mer duplex was used as a substrate, cleavage sites were observed on both strands. The location of the cleavage sites led us to conclude that the octathymidylate was bound to the (dA)8.(dT)8 sequence in a parallel orientation with respect to the (dA)8-containing strand. This result reflects the ability of thymine to form two hydrogen bonds with an adenine already engaged in a Watson-Crick base pair. This study shows that it is possible to design DNA-binding oligodeoxynucleotides that could selectively recognize and cleave polypurine-polypyrimidine sequences in double-stranded DNA.     

2,3-Diaminopyrazines as rho kinase inhibitors

Inhibition of rho kinase (ROCK) has been recognized as an important target for a number of diseases, including glaucoma. Herein we report SAR development around two hits from a kinase library that led to the discovery of the ROCK inhibitor compound 38. In vitro and in vivo analysis of this compound, including its effects in a monkey model of glaucoma will be discussed.     

Characterization, chromosomal assignment, and role of LIFR in early embryogenesis and stem cell establishment of rabbits

Leukemia inhibitory factor (LIF) is a multifunctional cytokine with an important role during early embryonic development, implantation, and as an inhibitor of murine embryonic stem cell differentiation. It exerts its effects by binding to the leukemia inhibitory factor receptor, a heterodimer of two transmembrane proteins, the specific leukemia inhibitory factor receptor subunit, and the common gp130. A partial cDNA clone coding for the membrane-bound form of the specific rabbit leukemia inhibitory factor receptor was isolated from the genital ridge of 13.5 days postcoitum fetus. Fluorescent in situ hybridization analysis revealed that the rabbit leukemia inhibitory factor receptor gene is located on chromosome OCU11p11.1. It has been shown that the membrane-bound rabbit leukemia inhibitory factor receptor mRNA is expressed during embryo implantation but not at earlier developmental stages. Rabbit embryonic stem cell-like line establishment is improved in the presence of LIF, and those cells express both leukemia inhibitory factor and its receptor. The withdrawal of leukemia inhibitory factor results the differentiation of embryonic stem cell-like cells to beating myocardial-like cells. Our findings suggest that the self-renewal mechanism is similar in mouse and rabbit embryonic stem cells, and expands our knowledge on the role of the LIF-LIFR signal pathway in early rabbit embryogenesis and rabbit embryonic stem cell establishment.     

Titration of Human Coronaviruses Using an Immunoperoxidase Assay

Calculation of infectious viral titers represents a basic and essential experimental approach for virologists. Classical plaque assays cannot be used for viruses that do not cause significant cytopathic effects, which is the case for strains 229E and OC43 of human coronavirus (HCoV). An alternative indirect immunoperoxidase assay (IPA) is herein described for the detection and titration of these viruses. Susceptible cells are inoculated with serial logarithmic dilutions of samples in a 96-well plate. After viral growth, viral detection by IPA yields the infectious virus titer, expressed as "Tissue Culture Infectious Dose" (TCID50). This represents the dilution of a virus-containing sample at which half of a series of laboratory wells contain replicating virus. This technique is a reliable method for the titration of HCoV in biological samples (cells, tissues or fluids).Download video file.^{(92M, mov)}     

Spontaneous development of a pancreatic exocrine disease in CD28-deficient NOD mice

Autoimmune pancreatitis (AIP) is a heterogeneous autoimmune disease in humans characterized by a progressive lymphocytic and plasmacytic infiltrate in the exocrine pancreas. In this study, we report that regulatory T cell-deficient NOD.CD28KO mice spontaneously develop AIP that closely resembles the human disease. NOD mouse AIP was associated with severe periductal and parenchymal inflammation of the exocrine pancreas by CD4(+) T cells, CD8(+) T cells, and B cells. Spleen CD4(+) T cells were found to be both necessary and sufficient for the development of AIP. Autoantibodies and autoreactive T cells from affected mice recognized a approximately 50-kDa protein identified as pancreatic amylase. Importantly, administration of tolerogenic amylase-coupled fixed spleen cells significantly ameliorated disease severity, suggesting that this protein functions as a key autoantigen. The establishment and characterization of this spontaneous pancreatic amylase-specific AIP in regulatory T cell-deficient NOD.CD28KO mice provides an excellent model for the study of disease pathogenesis and development of new therapies for human autoimmune pancreatitis.